Screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy

ABSTRACT

The invention relates to assays for screening growth factors, adhesion molecules, immunostimulatory molecules, extracellular matrix components and other materials, alone or in combination, simultaneously or temporally, for the ability to induce directed differentiation of pluripotent and multipotent stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/322,612 filed Feb. 3, 2009, now issued as U.S. Pat. No.9,334,478; which is a continuation application of U.S. application Ser.No. 10/227,282 filed Aug. 26, 2002, now abandoned; which claims thebenefit under 35 USC §119(e) to U.S. Application Ser. No. 60/314,316filed Aug. 24, 2001, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods for the in vitro culture anddifferentiation of totipotent, nearly totipotent, and pluripotent cells,and cells derived therefrom. Examples of such cells are embryonic cells,embryonic stem cells, embryonic germ cells, embryoid bodies, inner cellmass cells, morula-derived cells-derived cells, non-embryonic stem cellsof embryonic, fetal, and adult animals, such as mesenchymal,hematopoietic, and neuronal stem cells, and cells derived from any ofthese.

In one aspect, the invention provides efficient, high-throughput assaysfor screening and identifying chemical and biological agents andphysical conditions that may be used to induce and direct thedifferentiation of totipotent, nearly totipotent, and pluripotent cells,and cells therefrom along particular developmental lineages. Examples ofsuch differentiation-inducing agents and conditions are growth factors,cytokines and extracellular matrix components, cell-cell interactions,environmental conditions (temperature, oxygen pressure, etc.), and otherextracellular factors or components, and combinations thereof, to whichthe target stem cells may be exposed simultaneously or sequentially toinduce and direct differentiation.

In another aspect, the invention provides a means of making geneticallymodified stem cell lines, e.g., gene trap stem cell lines, thatfacilitate the production, isolation, and therapeutic use ofdifferentiated cell types for cell therapy.

In another aspect, the invention provides a means of producing andisolating particular types of cells for animal testing and cell therapy.

In another aspect, the invention encompasses compositions of growthfactors, cytokines, and/or other chemical and biologicaldifferentiation-inducing agents, alone or in combination, that areidentified by the methods described herein, and their use to direct thedevelopment of characterized cell populations and tissues fromtotipotent, nearly totipotent, and pluripotent cells, and cellstherefrom, for use in treatments, transplantation therapies, and drugdiscovery, including the discovery of novel cancer targets andtherapies.

Background Information

The past decade has been characterized by significant advances in thescience of cloning, and has witnessed the birth of a cloned sheep, i.e.,“Dolly” (Roslin Bio-Med), a trio of cloned goats named “Mira” (GenzymeTransgenics) and over a dozen cloned cattle (Advanced Cell Technology orACT). Most recent additions to the clone family include pigs (PPLTherapeutics) and mice (University of Hawaii Medical School). Scientistsat ACT have also demonstrated successful cross-species nuclearreprogramming by the birth of a cloned guar produced using a bovinerecipient oocyte. For example, see U.S. patent application Ser. No.09/685,062, incorporated by reference herein in its entirety.Furthermore, cloning technology has also advanced such that a mammal maynow be cloned using the nucleus from an adult, differentiated cell,which scientists now know undergoes “reprogramming” when it isintroduced into an enucleated oocyte. See U.S. Pat. No. 5,945,577,incorporated herein by reference in its entirety.

The showing that an embryo and embryonic stem cells may be generatedusing the nucleus from an adult differentiated cell has excitingimplications for the fields of organ, cell and tissue transplantation.There are currently thousands of patients waiting for a suitable organdonor, who face the problems of both availability and incompatibility intheir wait for a transplant. By using a differentiated cell from apatient in need of a transplant to generate embryonic stem cells, andinducing these to differentiate into characterized populations of thecell type required in the transplant, the problem of transplantationrejection and the dangers of immunosuppressive drugs could be precluded.This prospect is now known to many as “therapeutic cloning,” or “adultcell reprogramming” so as to distinguish it from “reproductive cloning”and provides a moral boundary as the reach of cloning extends toward therealm of human beings. Lanza et al., September 1999, Human therapeuticcloning, Nat. Med. 5(9):975-7.

Conscious of the promise of therapeutic cloning, scientists are seekingto understand how to efficiently direct the differentiation oftotipotent and pluripotent stem cells into particular cell types andtissues, while at the same time deterring their differentiation intounwanted cells and tissues. Controlled, specific direction of celldifferentiation will come from deciphering the factors and signals thatcontrol embryonic development. The alternative, e.g., the randomdifferentiation of embryonic cells and subsequent dissection of desiredtissues, is both impractical and morally unacceptable for human therapy.

As used herein, a “stem cell” is a cell that has the ability to dividefor indefinite periods in culture and to give rise to daughter cells ofone or more specialized cell types.

As used herein, an “embryonic stem cell” (ES-cell) is a cell line withthe characteristics of the murine embryonic stem cells isolated frommorulae or blastocyst inner cell masses (as reported by Martin, G.,Proc. Natl. Acad. Sci. USA (1981) 78:7634-7638; and Evans, M. andKaufman, M., Nature (1981) 292:154-156) i.e., ES cells are immortal andcapable of differentiating into all of the specialized cell types of anorganism, including the three embryonic germ layers, all somatic celllineages, and the germ line.

As used herein, an “embryonic stem-like cell” (ES-like cell) is a cellof a cell line isolated from an animal inner cell mass or epiblast thathas a flattened morphology, prominent nucleoli, is immortal, and iscapable of differentiating into all somatic cell lineages, but whentransferred into another blastocyst typically does not contribute to thegerm line. An example in the primate “ES cell” reported by Thomson etal. (Proc. Natl. Acad. Sci. USA. (1995) 92:7844-7848).

As used herein, “inner cell mass-derived cells” (ICM-derived cells) arecells derived from isolated ICMs or morulae before they are passaged toestablish a continuous ES or ES-like cell line.

As used herein, an “embryonic germ cells” (EG cells) is a cell of a lineof cells obtained by culturing primordial germ cells in conditions thatcause them to proliferate and attain a state of differentiation similar,though not identical to embryonic stem cells. Examples are the murine EGcells reported by Matsui, et al., 1992, Cell 70:841-847 and Resnick etal., Nature. 359:550-551. EG cells can differentiate into embryoidbodies in vitro and form teratocarcinomas in vivo (Labosky et al.,Development (1994) 120:3197-3204). Immunohistochemical analysisdemonstrates that embryoids produced by EG cells contain differentiatedcells that are derivatives of all three embryonic germ layers (Shamblottet al., Proc. Nat. Acad. Sci. U.S.A. (1998) 95:13726-13731).

As used herein, a “totipotent” cell is a stem cell with the “totalpower” to differentiate into any cell type in the body, including thegerm line following exposure to stimuli like that normally occurring indevelopment. An example of such a cell is an ES cell, an EG cell, anICM-derived cell, or a cultured cell from the epiblast of a late-stageblastocyst.

As used herein, a “nearly totipotent cell” is a stem cell with the powerto differentiate into most or nearly all cell types in the bodyfollowing exposure to stimuli like that normally occurring indevelopment. An example of such a cell is an ES-like cell.

As used herein, a “pluripotent cell” is a stem cell that is capable ofdifferentiating into multiple somatic cell types, but not into most orall cell types. This would include by way of example, but not limitedto, mesenchymal stem cells that can differentiate into bone, cartilageand muscle; hemotopoietic stem cells that can differentiate into blood,endothelium, and myocardium; neuronal stem cells that can differentiateinto neurons and glia; and so on.

As used herein, “differentiation” refers to a progressive, transformingprocess whereby a cell acquires the biochemical and morphologicalproperties necessary to perform its specialized functions.

As used herein, a “marker” is a characteristic or feature of a cell thatis indicative of a particular cellular state. Typically, a marker is abiochemical entity that changes state in a detectable manner when thecell enters or leaves a particular state. For example, a marker may be aDNA sequence encoding a product that is detectable (e.g., a specificmRNA, or a fluorescent or antigenic protein) or has detectable activity(e.g., a protein conferring antibiotic resistance or a chromogenicenzyme such as lacZ). When copies of the marker DNA sequence arerandomly inserted into the genomic DNA of a cell, some copies may beinserted proximal to a promoter in the correct orientation and in-framesuch that activation of the promoter results in transcription of themarker DNA sequence and synthesis of the detectable product that itencodes. Detection of the marker then identifies the cell as one thatcontains the marker gene in a transcriptionally active genetic locus.The term “marker” as used herein may refer to a marker gene, or to amarker RNA or protein encoded by such a gene.

Directed Differentiation of Stem Cells

Totipotent and nearly totipotent embryo-derived stem cells can beinduced to differentiate into a wide variety of cell types, some ofwhich are needed for cell therapy. For example, Anderson et al.demonstrated that inner cell masses (ICM) and embryonic discs frombovine and porcine blastocysts will develop into teratomas containingdifferentiated cell types from ectodermal, mesodermal and endodermalorigins when transplanted under the kidney capsule of athymic mice.Animal Repro. Sci. 45:231-240 (1996). Thomson et al. reported thatprimate ES cells are capable of differentiating into trophoblast andderivatives of the three embryonic germ layers, and describetransplanting primate ES cells into muscles of immunodeficient mice togenerate teratomas that also contain cells of the three embryonic germlayers, including tissues resembling neural tube, embryonic ganglia,neurons, and astrocytes (APMIS (1998) 106(1):149-156). ES cells of mice(Lee et al., Nature Biotech. (2000) 18:675-679), cynomolgus monkeys(Macaca fascicularis) (Cibelli et al., Science (2002) 295:819), andhumans (Zhang et al., Nature Biotech. (2001) 19:1129-1133) can becultured in vitro to generate embryoids that contain cells of all threegerm layers, including neural precursor cells that test positive fornestin (an intermediate filament protein produced in the developingcentral nervous system and widely used as a marker for proliferatingneural progenitor cells in the nervous system). Pluripotent stem cellscan be isolated from ES and EG cell-derived teratomas and embryoids andexposed to conditions that induce them to differentiate into specificcell types that are useful for cell therapy. For example,nestin-positive neural stem cells isolated from human embryoids can becultured under conditions that induce their differentiation into thethree major cell types of the central nervous system (see Zhang et al.(2001) p. 1130).

The foregoing reports describe the derivation of precursor ordifferentiated cells that appear to arise randomly or spontaneously inembryoids and teratomas generated from totipotent ES and EG cells.Production of a characterized population of differentiated cells bythese methods therefore requires isolating the differentiated cells ofinterest, or their precursors, from other types of cells in an embryoidor teratoma. Presently, there is strong interest in identifyingchemical, biological, and physical agents or conditions that inducetotipotent or nearly totipotent cells such as ES and EG cells todifferentiate directly into the desired differentiated cells, in orderto develop efficient methods for producing characterized populations ofdifferentiated cells that are useful for cell therapy.

In U.S. Pat. No. 5,733,727, Field described plating murine ES cells ontouncoated petri dishes and culturing them in medium that is free ofleukemia inhibitory factor (LIF), an inhibitor of differentiation, togenerate patches of cardiomyocytes that exhibit spontaneous contractileactivity (col. 12, lines 63-67). Field also described a useful methodfor purifying cells induced to differentiate into a specific cell typefrom other types of cells present in the culture: the parental ES cellsare cotransfected with a pGK-HYG (hygromycin) plasmid and a plasmidcontaining a MHC-neo^(r) fusion gene—an α-cardiac myosin heavy chain(MHC) promoter operably linked to a neo^(r) gene that confers resistanceto neomycin. The pGK-HYG plasmid provides selection for transfectedcells, while the MHC-neo^(r) gene permits a second round of selection ofthe differentiated cells—incubation in the presence of G418 eliminatesnon-cardiomyocyte cells in which the MHC promoter is inactive (see col.12, lines 63-67). The disclosure of U.S. Pat. No. 5,733,727 isincorporated herein by reference in its entirety.

Schuldiner et al. described a systematic approach to analyzing thedifferentiation of ES-derived cells in response to different growthfactors. They cultured human ES cells to generate embryoids, dissociatedthe embryoids and cultured the cells as a monolayer in the presence ofone of eight different growth factors. The differentiation induced bythe growth factors was examined by monitoring changes in the cells'morphologies, and by RT-PCR (reverse transcription-polymerase chainreaction) analysis of the expression of a panel of 24 cell-specificgenes in the parental ES cells, embryoid cells, and the dissociatedembryoid cells cultured in the presence or the absence of one of theeight growth factors. Schuldiner et al. reported that each of the growthfactors appeared to induce expression of different subset of the 24marker genes that were analyzed; and that the growth factor-treatedcultures were relatively homogenous, often containing only one or twocell types, whereas the dissociated embryoid cells cultured in theabsence of a growth factor spontaneously differentiated into manydifferent types of colonies. The growth factors appeared to act more byinhibiting than by inducing the differentiation of specific cell types,and none of the growth factors tested directed a completely uniform andsingular differentiation of cells, and suggesting that direction offormation of specific cell types will require combinations of factorsincluding those that inhibit undesired pathways and those that inducedifferentiation of specific cell types (see Proc. Natl. Acad. Sci. USA(2000) 97(21):11307-12). Paquin et al. described culturing murine P19 EScells under conditions resulting in formation of aggregates of cells,some of which differentiated into beating cardiomyocytes (Proc. Nat.Acad. Sci. (2002) 99(14):9550-9555). Reubinoff et al. describedmanipulating the conditions in which human ES cells were cultured toinduce their differentiation directly into neural precursors that couldthen be induced to differentiate into derivatives of the three neurallineages, neuronal cells, glial cells, and astrocytes (NatureBiotechnology (2001) 19:1134-1139). Kelly et al. have shown that changesin gene expression in ES cells in response to retinoic acid are highlyreproducible (Mol. Reprod. Dev. (2000) 56(2):113-23), a result thatimplies that growth factor-directed differentiation of embryonic cellsis dependably reproducible.

Other groups have had success in using a negative approach to identifyfactors necessary for the differentiation of ES cells into certain celltypes. For instance, Henkel and colleagues reported that thetranscription factor PU.1 is essential for macrophage development fromembryonic stem cells by showing that ES cells containing a homozygousknockout of the PU.1 gene failed to differentiate into macrophages (seeHenkel et al., Blood (1996) 88(8):2917-26). Similarly, Dunn andcolleagues demonstrated that knockout embryoid bodies containing atargeted disruption of the phosphatidylinositol glycan class A (Pig-a)gene failed to develop secondary hematopoietic colonies and demonstrateda grossly aberrant morphology (see Dunn et al., Proc. Natl. Acad. Sci.USA (1996) 93(15):7938-43).

Directed differentiation has also been demonstrated successfully inpluripotent adult stem cells. For instance, U.S. Pat. No. 5,942,225 toBruder et al. describes the lineage-directed induction of humanmesenchymal stem cell differentiation by exposing such stem cells to abioactive factor or combination of factors effective to inducedifferentiation either ex vivo or in vivo. Mesenchymal stem cells aremore differentiated than embryonic stem cells and only differentiateinto lineages including osteogenic, chondrogenic, tendonogenic,ligamentogenic, myogenic, marrow stromagenic, adipogenic and dermogeniclineages. Similarly, U.S. Pat. No. 5,851,832 to Weiss et al. describesthe in vitro proliferation and differentiation of neural stem cellsfollowing exposure of the cells to various growth factors. Such stemcells are limited in their differentiation potential, producing onlyneurons and glial cells, including astrocytes and oligodendrocytes (seealso Brannen et al., Neuroreport (2000) 11(5):1123-8; Lillien et al.,Dev. (2000) 127:4993-5005).

The studies described above have shown that totipotent, nearlytotipotent, and pluripotent stem cells can be induced to differentiateinto specific cell types by manipulating the concentration of growthfactors and cytokines in the medium in which they are cultured. Otherexamples of growth factor-induced differentiation include induction ofstem cells to become macrophages, mast cells or neutrophils by IL-3(Wiles et al., Development (1991) 111:259-267); the direction of cellsto the erythroid lineage by IL-6 (Biesecker et al., Exp. Hematol. (1993)21:774-778); induction of neuronal differentiation by retinoic acid(Slager et al., Dev. Genet. (1993) 14:212-224; Bain et al., Dev. Biol.(195) 168:342-357); and induction of myogenesis by transforming growthfactor (Rohwedel et al., Dev. Biol. (1994) 164, 87-101). In the latterexamples, the inducing agents were not directly applied to ES cells orcells directly derived from the embryo, but rather to aggregates of EScells or to embryoids.

In addition to manipulating the concentration of growth factors andcytokines, totipotent and pluripotent stem cells may be induced todifferentiate into specific cell types by co-culturing them with cellsof a different type. For example, Kaufman et al. (U.S. Pat. No.6,280,718) showed that human ES cells differentiate into hematopoieticprecursor cells when cultured on a feeder cell layer of mammalianstromal cells (see col. 5, line 7, to col. 6, line 26). The disclosureof U.S. Pat. No. 6,280,718 is incorporated herein by reference in itsentirety. Similarly, Kawasaki et al. have induced the differentiation ofcynomolgus monkey ES cells into dopaminergic neurons and pigmentedepithelial cells by culturing them on a feeder layer of murine stromalcells (see Proc. Natl. Acad. Sci. USA (2002) 99(3):1580-85).

As shown by the reports described above, research groups' attempts toidentify the agents or conditions that induce the differentiation oftotipotent and pluripotent stem cells into specific cell types generallyinvolve exposing the stem cells to one or two solutions containing arelatively small number of growth factors or cytokines, and monitoringto see if the stem cells differentiate to acquire a morphology and/or toexpress a marker gene that is characteristic of a specific cell type.

At present, there is a need for a systematic, large-scale, screeningassay to efficiently identify the combinations of biological,biochemical, and physical agents or conditions that act, simultaneouslyor sequentially, to induce the differentiation of totipotent, nearlytotipotent, or pluripotent stem cells into a large number of different,specific cell types.

Also needed are means for efficiently identifying, analyzing andcharacterizing marker genes and gene products that specifically mark keyregulatory steps associated with the induction of differentiation ofsuch stem cells into each of the important specific cell types.

There is also a need for an efficient means for producing and purifyingcharacterized populations of differentiated cells that are suitable anduseful for cell therapy, and for testing these in animal models.

The present invention accomplishes these ends, without being limitedthereto.

Differentiation Pathways in Oncogenesis

Many molecular events in oncogenesis are a recapitulation or mutation ofevents that normally occur in differentiation. In this respect, in manycases oncogenesis reflects a reversal of terminal differentiationutilizing, at least in part, pathways used in normal development.Control of cell growth and differentiation by extracellular signalsoften involves growth factor binding to high affinity transmembranereceptors such as the receptor tyrosine kinases (RTKs) For example,Recently Sakamoto et al., 2001, (Oncol. Rep. 8:973-80) reported thatnerve growth factor and its low-affinity receptor p75NGFR play a role inbreast cancer, Gmyrek et al., 2001 (Am. J. Pathol. 159:579-90) describedthe role of hepatocyte growth factor/scatter factor (HGF/SF) that bindsthe Met receptor and promotes the differentiation of epithelial cells inprostate, kidney, and hepatocellular carcinoma, similarly, mutations inthe Ret receptor has been implicated in multiple endocrine neoplasias,the kit receptor in mastocytomas and gastrointestinal tumors, the Flt-3ligand that plays a role in hematopoietic differentiation has beenimplicated in neural crest-derived tumors (Timeus et al., 2001, Lab.Invest. 81:1025-1037), FGF-1 and -2 in pancreatic malignancy (El-Hariryet al., 2001, Br. J. Cancer, 84:1656-63), HB-EGF in colon cancer (Ito etal., 2001, Anticancer Res. 21:1391-4), Oncostatin M in breast cancer,Glypicans in breast cancer (Matsuda et al., 2001, Cancer Res.61:5562-9), and Yiu et al., 2001 (Am. J. Pathol. 159:609-22) describedthe role of the extracellular matrix component SPARC in the apoptosispathway in ovarian cancer. These only a few examples of the manyextracellular components that are important in the differentiation of aparticular cell type, and also play a role in cancer. Surprisingly, fewassays for antitumor agents, or assays for novel targets in cancertherapy have been based on the identification of factors influencingearly differentiation pathways. The present invention also providesmeans for efficiently screening many combinations of biological,biochemical, and physical agents or conditions to identify treatmentsthat may induce cancerous cells to undergo differentiation and inhibittheir proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph that shows primate Cyno-1FF ES-like cellsconditioned to grow on tissue culture dishes without feeder fibroblasts(10×).

FIG. 1B shows Cyno-1FF cells at a higher magnification, showing thetypical morphology of ES-like cells (40×).

FIG. 2 consists of Table 1, which identifies the factors added to eachof the wells of the duplicate 24-well plates of Example 2.

FIG. 3 is a photograph showing Cyno-1FF cells that were exposed to Flt-3ligand.

FIG. 4 shows mesoderm and cells with the morphology of nestin positiveneuronal stem cells obtained by culturing Cyno-1FF cells in the presenceof TGF beta-1.

FIG. 5 shows cells having the appearance of endodermal precursor cellsobtained by culturing Cyno-1FF cells in the presence of theextracellular matrix protein tenascin.

FIG. 6 shows Cyno-1FF cells exposed to a chimeric protein made from thereceptor for Tie-1 and an immunoglobulin Fc region.

FIG. 7 shows fibroblast-like connective tissue cells produced byculturing Cyno-1FF cells in the presence of BMP-2.

FIG. 8 consists of Table 2, which identifies the primers that were usedto detect expression of cell type-associated genes by RT-PCR, and theexpected sizes of the DNA fragments produced by the RT-PCR reactions.

FIGS. 9A-9D show examples of the results of RT-PCR analysis of cellsfrom four different wells, each containing a different inducing agent(see Example 2). FIGS. 9A-9D show photographs of the lanes ofelectrophoretic gels in which the DNA molecules produced by RT-PCR wereseparated, stained with ethidium bromide, and illuminated with uv light.

FIG. 10 shows the detection of desmin by ICC in Cyno-1FF cells exposedto a differentiation-inducing agent (see Example 3).

FIG. 11 shows the detection of nestin by ICC in Cyno-1FF cells exposedto a differentiation-inducing agent (see Example 3).

FIG. 12A and FIG. 12B are phase contrast photographs of the cells inwell #16 of Example 5 that were exposed to IL-1-alpha.

FIG. 12A shows the arrowhead pointing to a beating myocardial cell.

FIG. 12B shows the arrowhead pointing to an endothelial cell adjacent tomyocardial cells.

FIG. 13 consists of Table 3, which identifies the combinations ofputative differentiation-inducing agents added to the wells of the 24well plates in which murine ES cells were cultured as described inExample 6.

FIG. 14 shows the detection of desmin by ICC in murine ES cells culturedin TGF-beta-1 and FGF-4 for five days on type I collagen and humanplasma fibronectin (see Example 6).

FIG. 15 shows the detection of X-gal staining of cells of the murinegene trap ES cell line K18E2 that were cultured for five days on type Icollagen and human plasma fibronectin in the presence of TGF-beta-1 andFGF-4 (see Example 7). Detection of expression of the markerbeta-galactosidase gene in the gene trap ES cells indicates that thecells were induced to differentiate.

FIG. 16 shows the detection of beta-galactosidase by ICC (using antibodyto beta-galactosidase) in cells of murine gene trap ES cell line M7H7that were cultured for five days on type I collagen and human plasmafibronectin in the presence of TGF-beta-1 and FGF-4. Nuclei areco-visualized by DAPI staining.

FIG. 17 shows the detection of beta-galactosidase by ICC in cells ofmurine gene trap ES cell line K18E2 that were cultured for five days ontype I collagen and human plasma fibronectin in the presence of FGF-4.

FIG. 18 shows the presence of β-galactosidase in K18E2 cells that werecultured with FGF-4 and TGF-β1 on inducer fibroblasts for 5 days, thensub-cultured for an additional 5 days with FGF-4 and TGF-β1 alone.

FIG. 19 shows the presence of β-galactosidase in M7H7 cells that werecultured with FGF-4 and TGF-β1 on inducer fibroblasts for 5 days, thensub-cultured for an additional 5 days with FGF-4 and TGF-β1 alone.

FIG. 20 shows the presence of β-galactosidase in K18E2 cells that werecultured with FGF-4 and TGF-β1 in the absence of inducer fibroblasts,and then sub-cultured for 5 more days in the same conditions.

FIG. 21 shows the presence of β-galactosidase in M7H7 cells that werecultured with FGF-4 and TGF-β1 in the absence of inducer fibroblasts,and then sub-cultured for 5 more days in same conditions.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention is to provide a high-throughputscreening assay for efficiently identifying chemical, physical, andbiological agents and/or conditions, and combinations of such agentsand/or conditions, that induce or direct the differentiation oftotipotent, nearly totipotent, or pluripotent stem cells, and cellstherefrom into a large number of different, specific cell types,including cell types that are useful for cell therapy.

Another object of the present invention is to provide efficient meansfor identifying and characterizing biochemical markers in cells that areassociated with the series of regulatory steps or “nodes” in thebranching pathways by which totipotent, nearly totipotent, orpluripotent stem cells, and cells therefrom differentiate into a largenumber of different, specific cell types, including cell types that areuseful for cell therapy.

Another object of the present invention is to provide efficient meansfor producing totipotent, or pluripotent stem cells, and cells therefromthat are genetically modified to facilitate the production, isolation,and therapeutic use of differentiated cell types for cell therapy.

In one aspect, the invention includes assays for identifying chemicaland biological agents and physical conditions which may be used todirect the differentiation of totipotent, nearly totipotent, andpluripotent cells, and cells therefrom along a particular developmentallineage. Examples of such differentiation-inducing chemical andbiological agents and physical conditions are growth factors, cytokinesand extracellular matrix components, cell-cell interactions,environmental conditions (temperature, oxygen pressure, etc.), and otherextracellular factors or components, and combinations thereof, to whichthe target cells may be exposed simultaneously or sequentially. Examplesof biological agents that can be used as putativedifferentiation-inducing agents include living or dead cells of alltypes, as well as portions or fractions of any cells, includingcompositions comprising organelles, internal and external cellmembranes, membrane-associated proteins, soluble proteins, proteincomplexes, complexes of proteins and other molecular classes, includinglipids, carbohydrates, and nucleic acids, etc. Methods for fractionatingcells to prepare fractions that may be used as biological agents thatare putative differentiation-inducing agents are well known. Otherbiological agents useful as differentiation-inducing agents are cellculture-conditioned medium, and extracts or fractions of natural orartificial tissues.

In another aspect, the invention provides means of making gene trap stemcell lines that have DNA encoding a detectable marker inserted as amarker gene in a genetic locus that is activated when the cellsdifferentiate. The DNA encoding the gene trap marker may be insertedin-frame with correct orientation at a site such that it is expressedand the marker is produced when the genetic locus in which it isinserted is activated. The inserted coding sequence then operates as amarker permitting detection of the differentiation of the stem cells.DNA encoding beta-galactosidase is an example of a commonly used genetrap marker suitable for the invention.

Another aspect of the present invention to provide efficient means forproducing totipotent, or pluripotent stem cells, and cells therefromthat are genetically modified to facilitate the production, isolation,and therapeutic use of differentiated cell types for cell therapy.

In another aspect, the invention provides a means of isolatingparticular types of cells for animal testing and cell therapy.

In another aspect, the invention encompasses compositions of growthfactors, cytokines, and/or other differentiation-inducing agents, aloneor in combination, that are identified by the methods described herein,and their use to direct the development of characterized cellpopulations and tissues from totipotent, nearly totipotent, andpluripotent cells, and cells therefrom, for use in treatments,transplantation therapies, and drug discovery, including the discoveryof novel cancer targets and therapies.

Nuclear transfer is a useful method for generating totipotent, nearlytotipotent, or pluripotent stem cells that can be used in the methods ofthe invention for screening agents and conditions that induce and directstem cells differentiation. The nuclear transfer methods useful forgenerating stem cells for the screening methods of the present inventionare the same as those for generating totipotent, nearly totipotent, orpluripotent stem cells that differentiate into cells that are useful forcell therapy. Such methods are described in the co-pending InternationalApplication filed on Jul. 18, 2002, based on U.S. ProvisionalApplication No. 60/305,904 and assigned to Advanced Cell Technology, thecontents of which are incorporated herein in their entirety, nucleartransfer can also be used to generate.

Stem Cells

The assays of the invention may be performed with any appropriatetotipotent, nearly totipotent, or pluripotent stem cells, and cellstherefrom. Such cells include inner cell mass (ICM) cells, embryonicstem (ES) cells, embryonic germ (EG) cells, embryos consisting of one ormore cells, embryoid body (embryoid) cells, morula-derived cells, aswell as multipotent partially differentiated embryonic stem cells takenfrom later in the embryonic development process, and also adult stemcells, including but not limited to nestin positive neural stem cells,mesenchymal stem cells, hematopoietic stem cells, pancreatic stem cells,marrow stromal stem cells, endothelial progenitor cells (EPCs), bonemarrow stem cells, epidermal stem cells, hepatic stem cells and otherlineage committed adult progenitor cells.

Totipotent, nearly totipotent, or pluripotent stem cells, and cellstherefrom, for use in the present invention can be obtained from anysource of such cells. One means for producing totipotent, nearlytotipotent, or pluripotent stem cells, and cells therefrom, for use inthe present invention is via nuclear transfer into a suitable recipientcell as described in U.S. Ser. No. 09/655,815, the disclosure of whichis incorporated herein by reference in its entirety. Nuclear transferusing an adult differentiated cell as a nucleus donor facilitates therecovery of transfected and genetically modified stem cells as startingmaterials for the present invention, since adult cells are often morereadily transfected than embryonic cells.

The methods of the invention may be performed with totipotent, nearlytotipotent, or pluripotent stem cells, and cells therefrom, of anyanimal species, including but not limited to human and non-human primatecells, ungulate cells, rodent cells, and lagomorph cells. Primate cellswith which the invention may be performed include but are not limited tocells of humans, chimpanzees, baboons, cynomolgus monkeys, and any otherNew or Old World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Rabbits are an example of alagomorph species with which the invention may be performed.

For example, the methods of the invention may be performed with murineES cells lines, or with primate ES or EG cell lines. An example of aprimate stem cell line with which the methods of the invention may beperformed is the totipotent non-human primate stem cell line Cyno-1,which was isolated from the inner cell mass of parthenogeneticCynomologous monkey embryos and is capable of differentiating into allthe cell types of the body. Cibelli et al. (Science (2002) 295:819).

Genetic Modification of Stem Cells

Some embodiments of the invention use stem cells that have beengenetically modified, or a library of such stem cells. For example,screening to identify agents or conditions that induce stem cells todifferentiate into a large number of different, specific cell types canbe carried out efficiently in a high-throughput manner using gene trapstem cell libraries, as discussed below.

After employing the screening assays of the invention to identify agentsor conditions that induce stem cells to differentiate into desired celltypes, e.g., cells that are useful for cell therapy, it is an aspect ofthe present invention to genetically modify the stem cells (either thegene trap cells, or unmodified ES cells of the same type), to facilitatethe production, isolation, and therapeutic use of differentiated celltypes for cell therapy.

For example, stem cells that give rise to differentiated cells for celltherapy can be genetically modified by correcting congenital mutations,or by introducing, altering, or deleting one or more genomic DNAsequences to provide therapeutic benefit to the patient receiving thecell transplant (gene therapy).

Nuclear transfer using an adult differentiated cell as a nucleus donorfacilitates the recovery of transfected and genetically modified stemcells as starting materials for the present invention, since adult cellsare often more readily transfected than embryonic cells.

In some instances, these cells may be genetically modified to express aselectable marker, or engineered with a genetic modification thatrenders the cells lineage defective. For instance, selectable markersmay be utilized to further purify specific cell types from samples ofdifferentiated cells derived using the methods reported herein. Suchmethods would include the use of positive selection wherein theselectable marker is, for example, the neomycin or hygromycin resistancegene. This allows the cells that have not differentiated into the chosencell type to be killed by G418 in the case of neomycin resistance.Alternatively, the specific promoter may drive other selection systemssuch as a cell surface antigen that allows, for instance, the isolationof the chosen cells using flow cytometry. Alternatively, cells may bemodified with a suicide gene operably expressed from a tissue-specificor lineage specific promoter, i.e., as a supplement to the compounds andcombinations identified using the methods disclosed herein, in order tofacilitate the recovery of desirable cells and tissues.

Culturing on Serum-Free Medium

Embryonic cells have the propensity to differentiate randomly andrapidly upon removal of LIF (leukemia inhibitory factor), and the feedercells normally used to maintain embryonic cells may produce growthfactors or other compounds that could complicate results (see Reubinoffet al., Nature Biotech. (2000) 18(4):399-404). Thus, an embodiment ofthe screening assays may include adapting the cells to a serum-freemedium or, in the case of some embryo-derived cells, to growth in theabsence of a fibroblast feeder layer in which they do not necessarilyneed to proliferate, but in which they will survive and remainresponsive to the test compounds applied. Different serum-free media areknown in the art and may be tested and used with any given cell line inthe methods disclosed herein. For instance, in evaluating the in vitrogrowth and differentiation of multipotent stem cells, U.S. Pat. No.5,851,832 (herein incorporated by reference) describes the use of aserum-free medium composed of DMEM/F-12 (1:1) including glucose (0.6%),glutamine (2 μM), sodium bicarbonate (3 mM), and HEPES. A definedhormone and salt mixture was used in place of serum. Wiles et al.describe a serum-free chemically defined medium (CDM) for studying EScell differentiation that fails to support spontaneous differentiationof ES cells while still permitting the evaluation of differentiation inresponse to exogenous factors (see Wiles et al., Exp. Cell Res. (1999)247(1):241-8). According to this group, in the absence of LIF and afeeder layer, ES cells typically differentiate rapidly, formingpredominantly endoderm, mesoderm and hematopoietic cells. However, inCDM, the cells still lose their ES cell phenotype but fail to formmesoderm. Rather, the cells enter a neuroectoderm commitment up to alimited point that is thought to be a type of “default” pathway thatoccurs in the absence of any exogenous differentiation signals.

Nichols and colleagues report the maintenance of ES cells in the absenceof a feeder layer with a combination of IL-6 plus soluble IL-6 receptor.Nichols et al., 1994, Derivation of germ-line competent embryonic stemcells with a combination of interleukin-6 and soluble interleukin 6receptor, Exp. Cell Res. 215(1):237-9. However, this combinationactivates the same signaling processes as does LIF, so this medium maynot be suitable to study the putative differentiation inducing factors.Although, it has been reported that ES cells do differentiate in thepresence of LIF (see Shen et al., Proc. Natl. Acad. Sci. USA (1992)89:8240-44). Furthermore, in vivo, LIF is present at the blastocyststage of development (Murray et al., Mol. Cell. Biol. (1990)10:4953-56). Thus, the response of ICM cells toward LIF may be regulatedtemporally and/or spatially in order to permit development to proceed.

Another group has isolated an ES cell line that is feedercell-independent and LIF-independent, and yet still contributes to allembryonic germ layers when placed in the environment of a developingembryo (Berger et al., Growth Factors (1997) 14(2-3):145-59). However,the cells were isolated by selection through passage so the mutationsthat contribute to this self-renewal ability are not known.Nevertheless, one can isolate a similar line of ES cells to be used inthe present invention as an alternative to developing a specificmaintenance medium.

Another option is to maintain the embryonic cells on a feeder layer inthe presence of LIF until the time of the assay. In their evaluation ofthe effects of eight different growth factors on ES cells, Schuldinerand colleagues transferred the ES cells to gelatin coated plates forfive days to allow for initial differentiation as aggregates, thenreplated the cells as a monolayer wherein the cells were exposed to thetest growth factors. See Schuldiner et al., 2000, supra. A similarapproach is commonly used to direct mouse ES cells in to specific celltypes, such as nerve cells or muscle cells (Slager et al., 1993, supra;Bain et al., 1995, supra; and Rohwedel et al., 1994, supra). However,Schuldiner also reported that the cells spontaneously differentiatedinto all different cell types in the absence of any tested growthfactor, wherein the samples that were treated with specific growthfactors were more homogenous than the untreated control. Thus, it may befor any particular assay that the combination of compounds tested willachieve the directed differentiation desired in the absence of specificmedia formulations that seek to deter differentiation. Indeed,researchers are finding that the process of directed differentiation mayinvolve compounds that inhibit certain developmental pathways eitheralone or in combination with inductive compounds.

Inducers of Differentiation

The methods of the invention may be used to screen a wide variety ofcompounds and culture conditions to determine their effect on thedifferentiation of stem cells. For instance, the methods may beperformed with one or more putative differentiation-inducing compoundsselected from the group consisting of growth factors, cytokines, factorsinvolved in cell-to-cell interactions, adhesion molecules, extracellularmatrix components, media components, environmental conditions, etc.Media components suitable for use include both identified andunidentified media components; for example, unidentified present inmedium conditioned by cell culture may be used as an inducer ofdifferentiation. The present invention includes screening to identifybiological compositions that comprise one or more unidentified agentsthat induce differentiation, and using known fractionation and assaymethods to isolate the active agent(s).

The methods and assays of the present invention may also be used toanalyze the differentiation of cells in response to materials isolatedfrom early stage fetuses or factors or homogenates or isolateddifferentiated cells derived therefrom. Other cells, including primarycells and tissues or isolated cell lines, may also be screened for theirpotential to induce the differentiation of cells according to thedisclosed methods and assays.

Examples of growth factors, chemokines, and cytokines that may be testedin the disclosed assays include but are not limited to the FibroblastGrowth Factor family of proteins (FGF1-23) including but not limited toFGF basic (146 aa) and it's variants, FGF acidic, the TGF beta family ofproteins including but not limited to TGF-beta 1, TGF-beta 2, TGF-betasRII, Latent TGF-beta, the Tumor necrosis factor (TNF) superfamily(TNFSF) including but not limited to TNFSF1-18, including TNF-alpha,TNF-beta, the insulin-like growth factor family including but notlimited to IGF-1 and their binding proteins including but not limited toIGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, the matrix metalloproteinasesincluding but not limited to MMP-1, CF, MMP-2, CF, MMP-2(NSA-expressed), CF, MMP-7, MMp-8, MMP-10, MMP-9, TIMP-1, CF, TIMP-2 andother growth factors and cytokines including but not limited to PDGF,Flt-3 ligand, Fas Ligand, B7-1(CD80), B7-2(CD86), DR6, IL-13 R alpha,IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, GDNF,G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sRbeta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR,beta-ECGF, TGF-alpha, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF,LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF,CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF,Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4,I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3,Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma,Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF,Oncostatin M, HRG-alpha (EGF Domain), TGF-beta 2, CNTF R alpha, Tie-2/FcChimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Flt-1), PDGF sR alpha,HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22,Activin A, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1(DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEMA/EGFR2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19,Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9,NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5,IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D,Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1delta/LKN-1/CCL15 (68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, ActivinRIA, EphA1, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc),Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta (IIIb), DNAM-1,Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma RI, IGFBP-2,IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB,GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), ActivinAB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L,BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin(OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 Rbeta 1, Dtk, LBP, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin(CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1(CD106), CD31 (PECAM-1), hedgehog family of proteins, Interleukin-10,Epidermal Growth Factor, Heregulin, HER4, Heparin Binding EpidermalGrowth Factor, bFGF, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, leptin,Interferon A, Interferon A/D, Interferon B, Interferon InducibleProtein-10, Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10,Cytokine Induced Neutrophil Chemoattractant 2, Cytokine InducedNeutrophil Chemoattractant 2B, Cytokine Induced NeutrophilChemoattractant 1, Cytokine Responsive Gene-2, and any fragment thereofand their neutralizing antibodies. The dosage can be in the range ofwell-established effective concentrations; for example, dosage can be inthe range of 0.1 to 5 times the maximum value of the EC₅₀, theconcentration that provokes a response halfway between baseline andmaximum.

Factors involved in cell-cell interactions that may be tested includebut are not limited to the ADAM (A Disintegrin and Metalloproteinase)family of proteins including ADAM 1,2,3A, 3B, 4-31 and TS1-9, ADAMTSs(ADAMs with thrombospondin motifs), Reprolysins, metzincins, zincins,and zinc metalloproteinases and their neutralizing antibodies.

Adhesion molecules that may be tested include but are not limited to Igsuperfamily CAM's, integrins, cadherins, including E-, P-, andN-cadherin, and selectins, and their neutralizing antibodies.

Nucleic acids that may be tested include but are not limited to thosethat encode or block by antisense, ribozyme activity, or RNAinterference transcription factors that are involved in regulating geneexpression during differentiation, genes for growth factors, cytokines,and extracellular matrix components, or other molecular activities thatregulate differentiation.

Extracellular matrix component may also induce and direct thedifferentiation of stem cells. Members of the tenascin family areexamples of extracellular matrix components that are useful in directingcell differentiation. There are currently five members of the family,tenascin-C (simply called tenascin in the examples below), andtenascins-R, -X, -Y and -W. Tenascin-R is especially useful in screensfor agents that induce cells of the central nervous system, whiletenascins-X and -Y are useful in screens relating to muscle cells.Tenascin-C is useful in differentiating a wide array of cell types,including neuronal and endodermal cells. Agents that block the action ofthe tenascins, such as neutralizing antibodies, and proteolytic subunitsof the tenascins are also useful in directing differentiation. Thetenascins or their subunits may be added to the culture substrate priorto the culture of the cells of interest, added to the media of thecultured cells, expressed by cells co-cultured with the cells ofinterest, or otherwise introduced into contact with the cells.

Extracellular matrix components that may be tested include but are notlimited to Tenascins, Keratin Sulphate Proteoglycan, Laminin, Merosin(Iaminin a2-chain), Chondroitin Sulphate A, SPARC, beta amyloidprecursor protein, beta amyloid, presenilin 1,2, apolipoprotein E,thrombospondin-1,2, heparin, Heparan Sulphate, Heparan sulphateproteoglycan, Matrigel, Aggregan, Biglycan, Poly-L-Ornithine, thecollagen family including but not limited to Collagen I-IV,Poly-D-Lysine, Ecistatin (viper venom), Flavoridin (viper venom),Kistrin (viper venom), Vitronectin, Superfibronectin, FibronectinAdhesion-Promoting peptide, Fibronectin Fragment III-C, FibronectinFragment-30KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45KDA,Fibronectin Fragment 70 KDA, Asialoganglioside-GM,Disialoganglioside-GOLA, Monosialo Ganglioside-GM₁,Monosialoganglioside-GM₂, Monosialoganglioside-GM₃, Methylcellulose,Keratin Sulphate Proteoglycam, Laminin and Chondroitin Sulphate A.Extracellular matrix components can be applied to the culture wellsprior to or after adding the cells. When coating the well surfaces, theconcentration of these components can be in the range of from 1 to 10mg/ml, or from 0.2 to 50 mg/ml.

Media components that may be tested include but are not limited toglucose concentration, lipids, transferrin, B-Cyclodextrin,Prostaglandin F₂, Somatostatin, Thyrotropin Releasing Hormone,L-Thyroxine, 3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Fetuin,Heparin, 2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, GoatSerum, Rabbit Serum, Human Serum, Pituitary Extract, Stromal CellFactor, Conditioned Medium, Hybridoma Medium, d-Aldosterone,Dexamethasone, DHT, B-Estradiol, Glucagon, Insulin, Progesterone,Prostaglandin-D₂, Prostaglandin-E₁, Prostaglandin-E₂, Prostaglandin-F₂,Serum-Free Medium, Endothelial Cell Growth Supplement, Gene TherapyMedium, MDBK-GM Medium, QBSF-S1, Endothelial Medium, KeratinocyteMedium, Melanocyte Medium, Gly-His-Lys, soluble factors that inhibit orinterfere with intracellular enzymes or other molecules including butnot limited to compounds that alter chromatin modifying enzymes such ashistone deacetylases such as butyrate or trichostatin A, compounds thatmodulates cAMP, protein kinanse inhibitors, compounds that alterintracellular calcium concentration, compounds that modulatephosphatidylinositol.

Environmental conditions that may be tested include but are not limitedto oxygen tension, carbon dioxide tension, nitric oxide tension,temperature, pH, mechanical stress, altered culture substrates such astwo vs. three dimensional substrates, growth on beads, inside cylinders,or porous substrates.

Materials derived from early stage embryos, fetuses, or adult tissuesthat may be tested include but are not limited to acellularextracellular matrix prepared by the detergent extraction of tissue fromembryoid bodies, primitive endoderm, mesoderm, and ectoderm, and theanlagen of differentiating organs and tissues or living cells or tissuesthat when co-cultured with the subject cells cause an induction ofdifferentiation.

Growth factors, adhesion factors, extracellular matrix components, etc.may be tested individually or in various combinations. In addition,these factors may be combined with various culture conditions, e.g.,vitamins and minerals, which may also have an effect on thedifferentiation of stem cells. For instance, it has been shown thatoxygen tension may influence gene expression and development in embryoidbodies. Bichet et al., 1999, Oxygen tension modulates β-globin switchingin embryoid bodies, FASEB J., 13:285-95. In assay formats that exposetest cells to a variety of different combinations, care should be takento document the conditions applied to each sample so that results may becorrelated to the appropriate test conditions.

Growth factors and other compounds may be applied to stem cells at about0.1 to about 10 times their effective concentration; for example, atabout 2 times their effective concentration, for varying periods oftime, e.g., one hour to two months depending on the timing ofdifferentiation of the cell of interest during normal development.Growth factors and other compounds can also be applied repetitively orin a particular temporal order with other compounds rather thansimultaneously, with hours, days or weeks passing between differentadministrations.

Screening Assays

An embodiment of the present invention uses a screening assay toidentify agents or conditions that induce the differentiation oftotipotent, nearly totipotent, or pluripotent stem cells, or cellstherefrom; e.g., cells selected from the group consisting of embryonicstem cells, embryonic germ cells, embryoid bodies, ICM cells,morula-derived cells, non-ES stem cells, and cells therefrom, and tocharacterize the type and degree of differentiation that occurs inresponse to the agents or conditions tested. For example, a screeningassay of the invention can comprise:

-   -   (a) separating individual totipotent, nearly totipotent, or        pluripotent stem cells, or cells therefrom, or groups of        individual cells, into one or a plurality of separate vessels        which may be open or closed, which vessels may be in the same or        different apparatus;    -   (b) isolating primary and/or progenitor cells from reference        tissues and placing said primary and/or progenitor cells into        separate vessels of a microarray thereby forming a control        reference library;    -   (c) exposing said separate vessels of totipotent, nearly        totipotent, or pluripotent stem cells, or cells therefrom, to        the same one or more putative differentiation-inducing compounds        either simultaneously or sequentially; and    -   (d) comparing said individual totipotent, nearly totipotent, or        pluripotent stem cells, or cells therefrom, or groups of cells,        to said reference library in order to evaluate the        differentiation of said individual cells or groups of cells.

High-Throughput Screening

A useful aspect of the present invention is that it provides means forscreening a large number of different types of stem cells; e.g., alibrary of gene trap stem cells selected to have gene trap markers thatare activated when the stem cells are induced to differentiate to alarge number of different steps or “nodes” in the complex, branchingtree of possible differentiation pathways leading to the partially orfully differentiated cell types of an animal. Moreover, the presentinvention also provides screening methods whereby one or more differenttypes of stem cells are exposed to a large number of different types ofchemical and biological agents and physical conditions, alone or incombination, simultaneously or in various temporal combinations, toidentify sets of agents and conditions that induce the stem cells topartially or fully differentiate into cell types of interest.

In performing the assays of the invention disclosed herein, individualcells or individual groups of cells may be separated into any type ofarray apparatus or assembly of compartments that is convenient forsystematically applying the test compounds and evaluatingdifferentiation. For instance, the cells may be distributed into anapparatus comprising 10 to 100,000 different vessels or compartments, orfor some embodiments 100 to 100,000 compartments, or for others 1000 to10,000 compartments, or separate wells of one or more multi-well plates.The multi-well plates that are used can have any number of wells; forexample, the screening assays of the invention can be performed using24- or 96-well plates. In this embodiment, the reference library ofprimary cells may be freshly isolated and distributed in a similar arrayapparatus, or alternatively, frozen stock cells may be used. Indistributing the cells into compartments, e.g., the wells of one or more24- or 96-well plates, 1 to 10⁶ stem cells can be added per cm² ofsurface. For example, the screening assays of the invention can beperformed by adding 10 to 10⁵ cells per cm² of surface. Some ES cellsrequire a minimum number of cells to survive, for such cells, 3 to 10⁶stem cells should be added per cm² of surface. Induction ofdifferentiation by a given set of conditions occurs with a statisticalprobability; therefore, the more cells per well, the greater thelikelihood that a cell in the cell will be induced to differentiate.

Reference Library Cells and Cell Type-Associated Markers

The primary and/or progenitor cells used for the reference library mayinclude any cells of interest, i.e., any cells for which the operator isinterested in identifying differentiation inducing compounds orcompositions, including but not limited to brain cells, heart cells,liver cells, skin cells, pancreatic cells, blood cells, reproductivecells, nerve cells, sensory cells, vascular cells, skeletal cells,immune cells, lung cells, muscle cells, kidney cells, etc. The referencelibrary cells are then used as an experimental control when testing theexposed stem cells for those that have differentiated into theparticular primary cells in the reference library. Functional assaysspecifically geared toward detecting each of the cells in the referencelibrary are performed on the treated or exposed stem cells to correlatedifferentiation with a particular cell type in the reference library.

For instance, depending on primary and secondary antibodies and otherligand reagents available and what is known about the molecular markersspecific for particular cell types, immunocytochemistry may be used totest treated stem cells for the expression of proteins that correlate tospecific cells in the library. Alternatively, RT-PCR may be used to testthe samples for particular gene transcripts. There are many knownmolecular markers of differentiation of cell types that are detectable,e.g., with specific antibodies or by RT-PCR; examples include E-, P-,and N-cadherins, keratin, chAT, tyrosine hydroxylase, gamma enolase,PDX, amylase, CD34, VEGFR, cardiacmyosin, collagen II, sex determiningregion Y, frizzled-3, GATA 6, brachyury, PU.1 (Spi-1), hepatocytenuclear factor-3, alpha-2 type XI colagen, hepatic lipase, nerve growthfactor, sonic hedgehog, hematopoietically expressed homeobox, enolase-2,keratin 19, osteoblast-specific factor 2, globin transcription factor 1,myogenic factor 3, myosin heavy polypeptide 2, dopamine transporter,CD34, human serum albumin, pancreatin amylase, insulin promoter factor,beta-globin, Oct 4, cardiac alpha-myosin heavy chain, cardiac myosinlight chain 1, fibroblast growth factor 5 (FGF-5), SOX-1,alpha-fetoprotein (AFP), EMX-2, engrailed-2 (En2), Hesx-1, Hox B1,Krox-20, Mush-1, Nkx-1, Nkx-2, Pax-3, Pax-6, nestin, and GAPDH (ahousekeeping gene, useful as a control marker).

Cells in the reference library should be tested simultaneously as apositive control, to ensure that a negative result is not the failure ofthe assay itself rather than the absence of the particular protein ortranscript. Functional assays could also be used to measure theproduction of enzymes or metabolites produced by the particularreference primary and/or progenitor cells, for instance by enzyme-linkedimmunosorbent assays (ELISA), high performance liquid chromatography(HPLC), Western blots, radioimmune assays, etc. For example,dopaminergic neurons could be tested for KCI induced dopamine release,β-cells for glucose dependent insulin release, cardiomyocytes forsynchronous contraction, hepatocytes for triacylglycerol production, toname of few examples.

A second embodiment of the invention involves a method for evaluatingthe differentiation of totipotent, nearly totipotent, or pluripotentstem cells, or cells therefrom; in response to different compounds orcombinations of compounds, comprising:

-   -   (a) separating individual totipotent, nearly totipotent, or        pluripotent stem cells, or cells therefrom, or groups of        individual cells, into one or a plurality of separate vessels        which may be open or closed, which vessels may be in the same or        different apparatus;    -   (b) systematically exposing said separate vessels of totipotent,        nearly totipotent, or pluripotent stem cells, or cells        therefrom, to a panel of different putative        differentiation-inducing compounds or combinations thereof        either simultaneously or sequentially; and    -   (c) comparing said individual totipotent, nearly totipotent, or        pluripotent stem cells, or cells therefrom, or groups of cells,        to a reference differentiated or partially differentiated cell        in order to evaluate the differentiation of said individual        cells or groups of cells.

This embodiment differs from the first embodiment described above inthat the cells are treated with a panel of different compounds andcombinations of compounds, and the results are compared with a singlereference control in order to identify particular conditions thatresulted in directed differentiation into that cell type.

Although any of the functional assays described above may be used toanalyze the results, this second embodiment is most amenable to the useof RNA expression profiles. For instance, expression profiles can begenerated anytime at any pace and used to form a library that catalogsthe RNA expression profiles according to what factors produced thespecific profiles. Then, the profiles may be compared at any time toexpression profiles from various reference primary cells in order tomatch each embryonic differentiation profile with a primary cell. Suchlibraries may be saved in electronic form, whereby matches in RNAexpression profiles as between the library members and any particularprimary or progenitor may be performed electronically rather than withthe naked eye.

A third embodiment involves a method for evaluating the differentiationof totipotent, nearly totipotent, or pluripotent stem cells, or cellstherefrom, in response to different compounds or combinations ofcompounds, comprising:

-   -   (a) isolating a transfected totipotent, nearly totipotent, or        pluripotent stem cell, or cell therefrom, wherein said cell is        transfected with at least one reporter gene, the expression of        which is operably linked to a promoter and/or gene of interest;    -   (b) expanding said transfected cell in culture;    -   (c) separating individual transfected cells or individual groups        of transfected cells into one or a plurality of separate vessels        which may be open or closed, which vessels may be in the same or        different apparatus;    -   (d) exposing said separate vessels of transfected cells to a        panel of different putative differentiation-inducing compounds        or combinations thereof either simultaneously or sequentially;        and    -   (e) analyzing said individual transfected cells or groups of        cells in order to detect expression of said at least one        reporter gene.

Alternatively, this embodiment may be performed using gene trap stemcells in which the marker DNAs are randomly inserted at sites such thatthey are expressed upon activation of genetic loci associated with thepartial or complete differentiation of the stem cells to a particularcell type. Such cells serve as functional markers of differentiation,even when the genetic loci into which the markers are inserted have notbeen identified.

In this embodiment, transfected totipotent, nearly totipotent, orpluripotent stem cells, or cells therefrom, are exposed to a panel ofdifferent compounds and combinations of compounds, in order to identifythose combinations that turn on expression of a particular reporter geneconstruct.

Stem cells comprising a relevant reporter gene constructs are known inthe art as discussed supra, or alternatively, can be produced accordingto known methods. For example, a reporter gene may be targeted to thelocus of a gene of interest, i.e., a gene specifically expressed in thecell or tissue type desired, by homologous recombination. By includingan internal ribosome entry sequence (IRES) and designing the vector suchthat insertion occurs downstream of the endogenous stop codon, thetranscript from the targeted locus will act as a bicistronic message,making both the endogenous protein and the protein encoded by thereporter gene. In this manner, the targeted gene will not befunctionally disrupted. Alternatively, the targeted integration may bedesigned such that a fusion transcript, and/or fusion protein results.

A second approach would be to construct reporter transgenes usingisolated promoter sequences for cell type specific genes. This approachis not as sophisticated since homologous recombination is not required,so it suffers from possible position variegation in transgeneexpression. However, the constructs may be made much more easily, andthe use of good 5′ and 3′ flanking sequences, and possibly insulatorsequences, could alleviate some of the variability.

The reporter gene strategy permits high-throughput and non-invasivescreening. Specifically, cells could be continuously monitored, so theassay point would not be restricted to any particular time period duringthe differentiation process. The screening can be performed conducted byplating transgenic stem cells onto 96 well plates, for instance, andsupplying each well with different conditions until reporter geneexpression is detected. This would enable different styles ofexperimental design to rapidly be employed and evaluated. Further, thisstrategy could also be coupled to other functional and morphologicalmarkers in the same cell population.

Using the reporter gene strategy, the activation of gene expressionspecific to certain cells types may be quantified with respect to thepurity of cells within the population. For example, the methods of theinvention could include the further steps of:

-   -   (f) quantitatively determining the amount of detectable signal;        and    -   (g) comparing said amount of detectable signal with the amount        of signal produced by the same number of said transfected cells        in the absence of any test compound.

This aspect could also facilitate development of compound combinationsthat yield purer cell populations. In addition, cells expressing areporter gene such as green fluorescence protein (GFP) may be purifiedfrom other cells or undifferentiated cells in the same sample byfluorescence activated cell sorting. Odorico et al., 2001, Multilineagedifferentiation from human embryonic stem cell lines, Stem Cells19(3):193-204.

Possible loci to be targeted for clinical applications are: insulin inβ-cells, DOPA decarboxylase in dopaminergic neurons, cardiac α-actin incardiomyocytes, and albumin in hepatocytes. Expression of these proteinsare absolutely restricted to the corresponding cell types, thus shouldprovide a reliable indicator or promoter source for the type of cellsbeing produced.

Reporter Genes Useful as Markers

Reporter genes useful for the present invention encode proteins that aredetectable by any means, i.e., those that are detectable by the nakedeye or after microscopic, photographic or radiographic analysis, orafter contacting said exposed cells with a reagent selected from thegroup consisting of chromogenic substrates, dyes, sugars, antibodies,ligands, primers, etc. Suitable reporter genes may encode polypeptidesincluding but not limited to green fluorescent protein (GFP), enhancedgreen fluoresent protein (EGFP), luciferase, chloramphenicalacetyltransferase, β-glucuronidase, β-galactosidase, neomycinphosphotransferase, alkaline phosphatase, guanine xanthinephosphoribosyltransferase or β-lactamase. See, e.g., U.S. Pat. No.5,928,88, herein incorporated by reference. The use of a marker geneencoding a fluorescent protein such as GFP permits detection ofexpression of the marker gene without injuring the cells. Fluorogenicsubstrates include but are not limited to fluoresceindi-β-D-galactopyranoside, resorufin β-D-galactopyranoside, DDAOgalactoside, methylumbelliferyl galactoside or its di-fluorinatedanalog, carboxyumbelliferyl galactoside, fluorescent glycolipids, AmplexRed Galactose, PFB Aminofluorescein, chloromethyl and lipophilicderivatives of DiFMUG, 4-methylumbelliferyl β-D-glucuronide, fluoresceindi β-D-glucuronide, 5-(pentafluorobenzoylamino)fluorescein diβ-D-glucuronide, DDAO β-D-glucuronide, etc. Those skilled in the art arefamiliar with many reagents for detecting glycosidase activity.

A fourth embodiment involves a method for evaluating the differentiationof transfected totipotent, nearly totipotent, or pluripotent stem cells,or cells therefrom, in response to one or more compounds, comprising:

-   -   (a) obtaining a library of transfected totipotent, nearly        totipotent, or pluripotent stem cells, or cells therefrom, each        transfected with at least one reporter gene, the expression of        which is operably linked to a pre-characterized promoter and/or        gene of interest;    -   (b) separating individual members of said library into one or a        plurality of separate vessels which may be open or closed, which        vessels may be in the same or different apparatus;    -   (c) exposing said separate vessels of transfected cells to the        same one or more putative differentiation-inducing compounds        either simultaneously or sequentially; and    -   (d) analyzing said individual members of said library in order        to detect expression of said at least one reporter gene.

This embodiment differs from the third embodiment described above inthat a library of different transfected cells, each comprising adifferent reporter construct is exposed to a single test compound ortest combination (rather than a panel of compounds being applied to asingle type of stem cell representing a single reporter construct).

As for the previous embodiment, this embodiment may be performed usinggene trap stem cells in which the marker DNAs are randomly inserted atsites such that they are expressed upon activation of genetic lociassociated with the partial or complete differentiation of the stemcells to a particular cell type, as such gene trap cells function asmarkers of differentiation, even when the genetic loci in which they areis inserted are unidentified.

The present invention also includes identifying agents and/or conditionsthat induce stem cell differentiation, and then genetically modifyingstem cells to facilitate isolation of a characterized population of thedifferentiated cells; e.g., to use in therapeutic trials in animalexperimental models. A non-limiting example of how this can be done isto transfect the stem cells with a marker DNA encoding a non-immunogeniccell surface antigen that is inserted into a genetic locus that isspecifically expressed in the differentiated cells to be isolated. Knownmethods, e.g., methods employing homologous recombination, can be usedto target the marker DNA to the desired locus. When the geneticallyaltered stem cells has differentiated into the desired cell type, themarker gene is expressed and the cells become tagged with the surfaceantigen. Methods for isolating genetically modified stem cells based onexpression of a marker protein such as a surface antigen are describedin Gay (U.S. Pat. No. 5,639,618), the contents of which are incorporatedherein by reference. In an embodiment of this aspect of the invention,an isolation marker is inserted into a stem cell to be expressed whenthe cell has differentiated into a precursor of several specific celltypes of interest. Additional isolation markers can be inserted into thesame cell for expression when the precursor cells terminallydifferentiate into specific cell types. This permits isolation bf eitherthe precursor cells, or the terminally differentiated cell types. Forexample, an isolation marker could be inserted into the locus of thenestin gene, a marker of neural precursor cells, to permit isolation ofneural precursor cells; and additional isolation markers can be insertedinto genetic loci that are specifically expressed in neuronal cells,glial cells, and astrocytes, to permit efficient isolation of thesecells types after induction of their differentiation from the neuralprecursor cells.

The present invention further includes identifying agents and/orconditions that induce stem cell differentiation, and then geneticallymodifying stem cells to constitutively express a marker gene thatpermits detection of differentiated cells derived from the geneticallymodified stem cells following their administration to an animal.

Accordingly, it is an embodiment of the invention to utilize cells froma species wherein a marker gene is used to identify a differentiatedcell of interest, and to transfect these cells with a constitutivelyexpressed marker such as Green Fluorescent Protein (GFP). Differentiatedcells resulting from these embryonic cells are useful in testing theefficacy and safety of cell in cell therapy in animal or human models.Expression of the cell type-associated marker demonstrates to theinvestigator that the cell type of interest is present in the targettissue of interest, and the constitutively expressed marker identifiesthe administered cells against the background of the host cells of theanimal into which the cells being tested were administered.

This embodiment may be performed by specifically preparing andcharacterizing a tailored panel of stem cells comprising a specific setof reporter constructs according to the techniques discussed above.Methods and materials for making and analyzing reporter gene constructsin eukaryotic cells, commonly called gene trap vectors, are known in theart and could be geared toward specific stem cells of interest onceappropriate vectors are identified. See, e.g., U.S. Pat. No. 5,922,601,herein incorporated by reference in its entirety; see also Salminen etal., 1998, Dev. Dyn. 212(2):326-33 and Stanford et al., 1998, Blood92(12):4622-31, each incorporated by reference in its entirety.

The members of any specially designed panel may be precharacterized orspecifically designed to be representative of a particular cell type orlineage. Procedures for preselecting and precharacterizing specific genetrap lines are known in the art. See Baker et al., 1997, Dev. Biol.185(2); Thorey et al., 1998, Mol. Cell. Biol. 18(5):3081-88; and Bonaldoet al., 1998, Exp. Cell Res. 244:125-36, each of which is incorporatedherein in their entirety. Alternatively, a panel of gene trap stem cellshaving random insertions may be accumulated, and the insertions thatrespond to a given compound or combination of compounds may becharacterized subsequently to exposure and identification. For instance,the location of the insertion may be identified by molecular cloningfollowing PCR of the flanking endogenous genetic material, and bysequencing outward from the inserted construct using well-establishedtechniques. See, e.g., Gossler et al., 1989, Science 244(4903):463-5,incorporated herein in its entirety.

A pluripotent cell that is particularly preferred for use in designingsuch a panel is the Cyno-1 cell line, a pluripotent primate stem cellline isolated from parthenogenetically activated oocytes fromCynomologous monkeys.

Screening with Pre-Existing ES Cell Gene Trap Libraries

In screening stem cells to determine agents and conditions that inducetheir differentiation to particular cell types, it is useful to use agene trap stem cell library comprised of stem cells in which the markergenes are inserted in genetic loci that are normally activated when thecells is induced to differentiate, and are under transcriptional controlof the endogenous promoters of the loci where they are inserted. Thisensures that expression of the marker genes is controlled by the sameregulatory signals (e.g., transcription factors and factors that alterchromatin structure) as the endogenous promoters of the loci where theyare inserted.

As an alternative to preparing an entirely novel gene trap library, anembodiment of the present invention employs any of the murine ES ceilgene trap libraries that are already known and available in the art.See, e.g., Cecconi & Meyer, 2000, FEBS Letts 480:63-71; see also Duricket al., 1999, Genome Res. 9(11):1019-25. For instance, the German GeneTrap Consortium (GGTC) has been established in Germany to provide areference library of gene trap sequence tags (GTST) in mouse embryonicstem cells. See Wiles et al., 2000, Nature Genetics 24(1), incorporatedby reference in its entirety. Sequence information on the GTST libraryis accessible at the Internet site of the GGTC, and the mutant ES cellsare freely available upon request to the scientific community. Anotherlibrary of gene trap murine ES cells, called OmniBank®, is alsoavailable from Lexicon Genetics, Inc. (The Woodlands, Tex.), who havereportedly characterized over 20,000 sequence-tagged mutations. TheOmniBank® database may be searched using keywords or nucleotide orprotein sequences via the Internet site of Lexicon Genetics, Inc. Seealso Zambrowicz & Friedrich, 1998, Int. J. Dev. Biol. 42(7):1025-36;Zambrowicz et al., 1998, Nature 392:609-11; see also U.S. Pat. Nos.6,080,576, 6,207,371 and 6,136,566, each herein incorporated byreference in their entirety. Another group reported the successfulrecovery of 115 sequences from 153 cell lines using 5′ RACE technology.Townley et al., 1997, Genome Res. 7:293-298, incorporated by referencein its entirety. Sequence information from some of these murine ES cellclones is available on the University of California/Berkeley web site ofthe Skarnes lab. In addition, details on a large number of otheracademic mouse ES cell tagging efforts have also recently been reported.Chowdhury et al., 1997, Nucleic Acids Res. 25:1531-1536; Hicks et al.,1997, Nat. Genet. 16:338-344; Couldrey et al., 1998, Dev. Dyn.212:284-292; and Voss et al., 1998, Dev. Dyn. 212:171-180, each of whichis incorporated by reference herein in its entirety.

Gene-trap ES cells have been used to generate large numbers of mutantorganisms for genetic analysis. The retrieval of transgenic mice madefrom gene trap ES cells has allowed for trapped genes to becharacterized and segregated based on tissue expression profile, orsubcellular expression characteristics. Some predict that genome-widegene-trapping strategies, which integrate gene discovery and expressionprofiling, can be applied in a parallel format to produce living assaysfor drug discovery. Durick et al., 1999, supra. The use of gene trapclones in in vitro studies, on the other hand, has been limited.

U.S. Pat. No. 6,080,576 to Zambrowicz suggests using gene trap ES cellsto screen for secreted molecules that induce apoptosis or hematopoieticcell differentiation. However, this approach is geared towardidentifying insertions that cause over-expression of endogenous genes,and does not provide a format for systematically screening large numbersof compounds for their effect on stem cell differentiation. Similarly,Russ and colleagues disclose the identification of genes induced byfactor deprivation in hematopoietic cells undergoing apoptosis usinggene trap mutagenesis. Russ et al., 1996, Identification of genesinduced by factor deprivation in hematopoietic cells undergoingapoptosis using gene-trap mutagenesis and site-specific recombination,Proc. Natl. Acad. Sci. USA 93:15279-84. However, this approach looks forgenes activated during programmed cell death rather than genes activatedduring embryonic or stem cell differentiation.

Era and colleagues utilize a LacZ reporter gene similar to that used ingene trap strategies in order to characterize hematopoieticlineage-specific gene expression by ES cells in an in vitrodifferentiation induction system. Era et al., 2000, Blood 95(3):870-78.However, this approach was geared toward analyzing a particular promoterof interest and determining which section of the promoter wasresponsible for differentiation-induced expression. There was nosuggestion to use the promoter constructs to screen for growth factorsor other compounds that are involved in particular cell lineagedifferentiation pathways.

Bonaldo and colleagues used gene-trap and pre-selection analysis ofisolated cell lines to identify fusions that are expressed duringembryonic development in response to specific, single growth factors.They do not use the cells identified, however, to screen forcombinations of factors that direct the development of those cells. Infact, the low-serum medium employed in the screening process was onlysuitable for short term screening lasting about 24 hours. See Bonaldo etal., 1998, supra. Thus, Bonaldo et al. presents a means of preselectingand precharacterizing cells containing fusions in the early developingembryo, but does not disclose the use of such cells in screening forfactors that direct the differentiation of specific cells and tissues.

Similarly, Forrester and colleagues used gene-trap technology toidentify genes specifically expressed in response to retinoic acidduring embryogenesis. Forrester et al., 1996, An induction gene trapscreen in embryonic stem cells: Identification of genes that respond toretinoic acid in vitro, Proc. Natl. Acad. Sci USA 93:1677-82. However,they also did not use such cells to screen for growth factorcombinations that direct the development of specific cells and tissues.

Thus, this is the first disclosure of which the present inventors areaware, that proposes to use the gene trap ES cell libraries as a toolfor screening growth factors, adhesion factors, extracellular matrixmaterials, etc., for compounds and combinations that mediate thedirected differentiation of stem cells. The ES cells identified ascorresponding to a specific combination of growth factors may be used tomake transgenic embryos or animals, in order to correlate in vivotemporal and spatial gene expression with the in vitro data obtained inthe disclosed method.

As indicated by the foregoing description, libraries of totipotentmurine and non-human gene trap stem cells can be assembled from existingcell lines, or novel libraries can be made using known techniques. Whengene trap marker DNAs are inserted randomly, developing or matureanimals cloned from the gene trap ES cells can be sacrificed andanalyzed histologically to identify the gene trap stem cell lines thatcontain markers that are activated in particular cell types for use inthe screening assays of the invention.

Alternatively, gene trap marker DNAs can be inserted into ES or EGcells, either directly or by deriving genetically modified ES or EGcells from a nuclear transfer embryo produced with a geneticallymodified nuclear donor cell. For example, a library of totipotent humangene trap stem cells can be produced by deriving a set of geneticallymodified human ES or EG cells from nuclear transfer embryos producedwith genetically modified nuclear donor cells. The gene trap ES or EGcells are then expanded and used to produce embryoids containing diversetypes of differentiated cells. Histological analysis is performed toidentify the gene trap stem cell lines that contain markers that areactivated in particular cell types for use in the screening assays ofthe invention. Alternatively, the totipotent cells can be injected intoan animal to produce teratomas containing diverse types ofdifferentiated cells, and histological analysis of these performed toidentify the gene trap stem cell lines that contain useful markers ofdifferentiation.

The present invention has broad applications. For example, in additionto identifying agents and conditions that induce and directdifferentiation, the screening methods of the present application permitidentification of agents and conditions that promote cell survival(survival factors), and of agents and conditions that promotemitogenesis (mitogenic factors). For example, cells are cultured for aperiod of 1-14 days with exposure to a panel of different agents andconditions that are putative survival and/or mitogenic factors, and theeffects of the various treatments on cell survival and/or cellproliferation over the time interval of the assay is determined. Agentsand conditions that decrease the loss or death of particular cell typescan be detected by the assay in this manner, and may be regarded assurvival factors. Similarly, agents and conditions that increase cellproliferation over the course of the assay are mitogenic factors. Thecombination of information regarding differentiation, survival, andmitogenic factors is useful in identifying and optimizing conditionsthat are useful for producing desired quantities of medically usefulcell types.

In another aspect, the invention encompasses compositions andformulations comprising the compounds and compositions identified usingthe disclosed methods, and the use thereof to direct the development ofcells and tissues from totipotent, nearly totipotent, and pluripotentstem cells, and cells therefrom, to isolate cells and tissues for use intreatments and transplantation therapies. In particular, the identifiedcombinations of factors may be used to induce the differentiation ofcells on polymeric matrices, i.e., as disclosed in U.S. Pat. Nos.6,214,369, 6,197,575, and 6,123,727, each of which is hereinincorporated by reference in its entirety.

The combinations identified by the disclosed methods may also be used toinduce the production of different types of cells, either separately orin conjunction, in order to design and recover tissues and/or artificialorgans constituted of different cell types.

Other embodiments, variations and modifications of the assays andmethods disclosed herein will be envisioned by those in the art uponreading the present disclosure, and should also be included as part ofthe invention.

EXAMPLE 1 Conditioning Totipotent Stem Cells to Grow and Maintain anUndifferentiated State in the Absence of Feeder Cells

ES-like cells derived from the inner cell mass of parthenogeneticCynomologous monkey embryos, Cyno-1 were originally cultured onmitotically inactivated mouse embryonic fibroblast derived from D12fetuses (strain 129).

The culture media was:

DMEM (High Glucose) (Gibco #11960-044) 425 ml Fetal Calf Serum (Hyclone)75 ml MEM non-essential AA x100 (Gibco #11140-050) 5 ml L-Glutamine 4 mM2-mercatoethanol (Gibco #21985-023) 1.4 ml

The cells were passaged mechanically every 4 to 5 days.

To condition the cells to grow in the absence of feeder cells to improvethe screening assay, the cells were passaged mechanically into anon-coated Polystyrene cell culture plate (Corning).

For the first two days, cells were cultured in conditioned media fromthe original cultures (described above).

On day three, conditioned media was replaced by:

-   -   Human Endothelial-SFM Basal Growth Medium (Gibco #11111-044) 500        ml    -   EGF-Human Recombinant (Gibco #10458-016) 10 μg    -   bFGF (Gibco #13256-029) 10 μg    -   Human Plasma Fibronectin (Gibco #33016-023) 1 mg

The colonies maintained their pluripotent phenotype (morphology and APstaining) for up to one week. The cultures appeared that grew in theabsence of feeder fibroblasts while maintaining an undifferentiatedstate. This new line designated Cyno-1FF displays the morphology ofundifferentiated ES-like cells in that they have small cytoplasmic tonuclear ratios, prominent nucleoli, and are alkaline phosphatasepositive (FIGS. 1A-1B).

EXAMPLE 2 Screen Using Primate ES-Like Cells and Analysis by Microscopyand RT-PCR

Approximately 10⁵ ES-like stem cells from parthenogenetic monkey embryos(Cyno-1FF cell line, see Example 1) were plated in duplicate 24 wellplates in the presence of mouse embryonic fibroblast-conditioned mediumfor two days. The media was then aspirated and replaced with DMEM mediumwith 15% fetal bovine serum, added nonessential amino acids, 5×10⁻⁵ Mβ-mercaptoethanol, 2 mM L-glutamine, 100 μg/ml penicillin, and 100 μg/mlstreptomycin. The cells were then cultured in the presence of growthfactors or cytokines in order to direct their differentiation. Workingstock solutions of the cytokines were prepared in 0.1% bovine serumalbumin (BSA) in phosphate-buffered saline (PBS). Diluted cytokines wereapplied on Day 0. To each well, 7.5 μl of diluted factor was added fromthe working stock solutions to obtain the following finalconcentrations:

-   -   VEGF-A (165 kDa) (R&D Biosystems cat# 293VE) was used at 20        ng/mL,    -   LAP (R&D #246-LP) at 50 ng/mL,    -   Flt-3/Flk-2 ligand (R&D #308-FK) at 5 ng/mL,    -   TGF beta-1 (R&D #240-B) at 0.1 ng/mL,    -   IGF-1 (R&D #291-G1) at 10 ng/mL,    -   PIGF (R&D #264-PG) at 20 ng/mL,    -   Tie-1/Fc chimera (R&D #619-TI) at 100 ng/mL,    -   BMP-2 (R&D #355-BM) at 500 ng/mL,    -   BMP-4 (R&D #314-BP) at 250 ng/mL,    -   BMP-5 (R&D #615-BM) at 2 μg/mL,    -   FGF-17 (R&D #319-FG) at 50 ng/mL,    -   TGF-alpha (R&D #239-A) at 0.5 ng/mL,    -   Fibronectin (human 120 chymotryptic fragment, Gibco #12159-018)        at 50 ng/mL,    -   Merosin (Gibco #12162-012) at 50 ng/mL,    -   Tenascin (Gibco #12175-014) at 50 ng/ml,    -   IL-1-alpha (R&D #200-LA) at 10 pg/mL,    -   FGF-4 (R&D #235-F4) at 0.25 ng/mL,    -   SCF (R&D #255-SC) at 10 ng/mL,    -   bFGF (R&D #233-FB) at 1.0 ng/mL,    -   PDGF (R&D #120-HD) at 5.0 ng/mL,    -   PECAM-1 (R&D #ADP6) at 1.0 /g/mL,    -   anti-FGF-4 antibody (R&D #AF235) at 0.5 μ/g/mL,    -   anti-Cripto-1 antibody (R&D #AF145) at 0.5 μ/g/mL,    -   and a control of the same volume of 0.1% BSA in PBS.

To coat a well with an ECM component, a solution of the ECM component ata concentration of 10 μg/mL in PBS was added to the well to be coatedand incubated for at least one hour, and then removed by aspiration.

The plates were cultured at 37 deg. C. at atmospheric O₂ and 5% CO₂, onefor three and one for ten days. Table 1 in FIG. 2 identifies the factorsthat were added to each of the wells of duplicate 24-well plates. Oneplate was harvested on Day 3, and the other plate was harvested on Day10. Analysis by phase contrast microscopy and RT-PCR revealed manyunique differentiated cell types, as discussed below.

Analysis of Cell Morphologies by Phase Contrast Microscopy

Following exposure to Flt-3 ligand, the Cyno-1FF ES-like cellsdifferentiated into cells that appeared to be vascular endothelial cells(derivatives of mesodermal differentiation). Cells having the appearanceof vascular endothelial cells were observed by five days in the wellswith added Flt-3 ligand, and were more evident in these wells by day 11.FIG. 3 is a photograph of primate Cyno-1FF cells exposed to Flt-3ligand.

Exposed to TGF beta-1 induced Cyno-1FF cells to acquire morphologiesthat appeared to be those of mesodermal and neural stem cells. FIG. 4shows mesoderm and cells with the morphology of nestin positive neuronalstem cells obtained by the culture of Cyno-1FF cells in the presence ofTGF beta-1.

Cyno-1FF cells cultured in the presence of the extracellular matrixprotein tenascin induced the formation of a distinctive population ofcells that had the appearance of endodermal precursor cells. Theappearance of the cells in the presence of tenascin was strikinglydifferent from that of the cells in the control well. This resultindicates that different concentrations of this particular extracellularmatrix component and/or its removal or inactivation can be used todirect the differentiation of totipotent and pluripotent stem cells.FIG. 5 shows cells with the appearance of endodermal precursor cellsobtained by the culture of Cyno-1FF cells in the presence of theextracellular matrix protein tenascin.

Cyno-1FF cells that were exposed to other putativedifferentiation-inducing agents in other wells of the assay plate werealso induced to differentiate to have distinctive morphologies and toexpress cell type-associated genes. For example, FIG. 6 shows theappearance of cells cultured in the presence of Tie-1 receptor/Fcchimera. Cells cultured in the presence of BMP-2 acquired the morphologyand appearance of connective tissue fibroblast-like cells, as shown inFIG. 7.

RT-PCR Analysis of Expression of Cell Type-Associated Genes:

The expression of cell type-associated genes by the Cyno-1FF cellsexposed to the panel of putative differentiation-inducing agents shownin FIG. 2 was assayed by RT-PCR using the following standard protocols.

-   -   (a) RNA was harvested from cells using kit: Ultraspec-II RNA,        Item No. BL-12050 (Bioflex Labs, Inc.) and included protocol.    -   (b) The isolated RNA was amplified using listed primers and kit:        Enhanced Avian RT First Strand Synthesis kit, Item No. STR-1        (Sigma-Aldrich, Inc.).    -   (c) The amplified RNA was harvested from cells and stored at        −70° C. in ethanol.    -   (d) Reverse transcription reaction:        -   RNA was resuspended to 30 ul, and the following reagents            were added:            -   2 ul dNTP mixture            -   2 ul Random nonamers        -   the mixture was heated to 80° C. for 12 minutes, then            transferred to an ice bath for 5 minutes        -   the following reagents were added:            -   4 ul 10× RT buffer            -   1 ul RNAse inhibitor            -   2 ul reverse transcriptase        -   the reaction was then thermocycled using the following            conditions:            -   24°—15 min            -   42°—50 min            -   95°—30 sec            -   4°—hold        -   the mixture was then aliquotted with 3 ul/sample and was            stored at −70° C. until use.    -   (e) Polymerase chain reaction:        -   The following reagents were added to each sample:            -   2 ul primer pair mix (50 pmol/ul)            -   5 ul MgCI2            -   PCR reaction mixture (for each sample):                -   5 ul 10× buffer (without Mg)                -   4 ul dNTP mix (10 mM)                -   0.5 ul Taq (Sigma)                -   0.055 ul HotStart Taq (Qiagen)                -   30.5 ul H2O        -   the reaction was then thermocycled for 35 cycles using the            following conditions:            -   94° C.—2 min            -   94° C.—30 sec            -   45° C.—1 min            -   72° C.—2 min            -   72° C.—10 min            -   4° C.—hold

The primers that were used to detect expression of cell type-associatedgenes by RT-PCR, and the expected sizes of the products, are shown inTable 2 shown in FIG. 8. The PCR products were visualized bypolyacrylamide gel electrophoresis, ethidium bromide staining, andillumination with uv light. The bands were identified by predicted sizeand relative intensity determined by comparison with GAPDH intensity.

Examples of the results, demonstrating detection of specificdifferentiation pathways in the endoderm, mesoderm, and ectoderm germlayers in the wells by RT-PCR, is shown in FIGS. 9A-9D.

FIGS. 9A-9D show that Cyno-1FF cells induced to differentiate bydifferent differentiation-inducing agents express different butsometimes overlapping combinations of cell type-associated genes. Forexample, cells exposed to VEGF-A expressed ChAT, keratin-19, and nestin,and cells exposed to tenascin expressed ChAT, nestin, and GATA-4. Thestrongest induction of ChAT (choline acetyltransferase) and therefore,of neuronal differentiation was seen in well 10-14 in the presence ofthe extracellular matrix component tenascin, and in well 10-15 in thepresence BMP-5. ChAT was also induced by TGF-beta-1, IGF-1, FGF-4, bFGF,tenascin, and anti-Cripto-1 antibody. The best endothelial/hematopoieticconditions observed were in the presence of Flt-3 ligand. Thiscorrelated well with the endothelial morphology observed by phasecontrast shown in FIG. 3. Interestingly, the best conditions observed toinduce endothelial differentiation were also in the presence of theextracellular matrix component tenascin.

In contrast to the results obtained with cells cultured in wellscontaining differentiation-inducing agents as described above,expression of cell type-associated genes by control Cyno-1FF cellscultured in medium without the added putative differentiation-inducingagents was no detected by the RT-PCR assay. This result is evidence thatthe above-described assay detected genuine differentiation-inducingeffects.

EXAMPLE 3 Screen Using Primate ES-Like Cells and Analysis byImmunocytochemistry

The presence of products of the expression of cell type-associated genesin Cyno-1FF cells exposed to putative differentiation-inducing agents inone of the 24 well plates prepared according to Example 2 was detectedby immunocytochemistry (ICC).

Solutions for Immunocytochemistry:

-   -   Fixative: 4% Paraformaldehyde    -   Permeabilization Solution: DPBS+1% TritonX-I00    -   Blocking Solution: DPBS+150 mM glycine+3 mg/ml BSA    -   Rinsing Solution: DPBS+0.1% Triton X-I00    -   Antibody Diluent: DPBS+0.1% Triton X-I00+3 mg/ml BSA

General Protocol for Immunocytochemistry:

-   -   Rinse cells in DPBS (with Ca/Mg so cells do not dissociate) 3×.    -   Add 4% Paraformaldehyde, Incubate at RT for 20-30 min.    -   Remove fixative with a Pasteur pipette and wash 3× with PBS. At        this point cells can be stored at 4 C for long periods of time        if wrapped in parafilm.    -   Add blocking solution and incubate at RT for at least 1 hour        (this can be prolonged as long as overnight).    -   Remove blocking solution and replace with primary antibody        (diluted . . . generally dilutions of 1:10 to 1:100 work well).    -   Incubate at RT for at least 1 hour.    -   Remove primary antibody and wash 3× with PBS over 45 minutes.    -   Add secondary antibody (diluted . . . generally dilutions of        1:50 to 1:500 work well).    -   Rinse 3× in PBS over 45 minutes. Add 5 ug/ml Hoechst or DAPI to        first rinse.    -   Sample is ready for imaging.

Antibodies Used:

-   -   GATA-4: Item # sc-1237 (Santa Cruz Biotechnology, Inc.)    -   Goat IgG used at dilution of 1:75    -   Nestin: Item# 611659 (BD Transduction Laboratories, Inc.)    -   Mouse IgG1 used at dilution of 1:75    -   Desmin: Item# D-1033 (Sigma-Aldrich, Inc.)    -   Mouse IgG1 used at dilution of 1:20    -   Goat anti-Mouse IgG-FITC conjugate: Item# F-0257 (Sigma-Aldrich,        Inc.)    -   Used at dilution of 1:50    -   Mouse anti-Goat IgG-FITC conjugate: ltem#sc-2356 (Santa Cruz        Biotechnology, Inc.)    -   Used at dilution of 1:50

The ICC assay successfully detected gene expression products associatedwith each of the three embryonic germ layers. FIG. 10 demonstrates thedetection by ICC of desmin, a marker for mesoderm, and FIG. 11demonstrates the detection of nestin, primarily a marker for ectoderm,but sometimes of endoderm, in Cyno-1FF cells exposed todifferentiation-inducing agents. The expression of GATA-4, a marker forendoderm, was also detected by ICC in Cyno-1FF cells exposed todifferentiation-inducing agents (results not shown).

EXAMPLE 4 Screen Using Primate ES Cells, Induction of Differentiation byPhysical Conditions

Cyno-1FF ES-like cells were plated in wells of a 24 well plate asdescribed in Example 2, and were incubated under low oxygen partialpressure (5%). A control plate of the same cells was incubated inambient oxygen. Analysis of cellular morphologies showed that the cellsincubated under low oxygen partial pressure (5%) were induced to acquiredifferent morphologies than the control cells incubated under ambientoxygen. This example demonstrates the importance of screening variousphysical as well as chemical factors to identify conditions or factorsthat induce differentiation of stem cells into desired cell types.

EXAMPLE 5 Screen for Agents that Induce Differentiation of Murine ESCells Into Myocardial Cells

Approximately 20,000 murine ES cells (strain J1) were plated in a 24well tissue culture plate without feeder fibroblasts or LIF in 1.5 mL ofDMEM Medium with 15% fetal bovine serum, added nonessential amino acids,5×10⁻⁵ M 2-mercaptoethanol, 2 mm L-glutamine, 100 ug/ml penicillin, and100 ug/ml streptomycin. The cells were incubated and allowed todifferentiate in the presence of the same added factors and in the samemanner as described in Example 2. After ten days of differentiation, themorphologies of the cells were examined by phase contrast microscopy todetect rhythmically contracting cells as evidence of myocardialdifferentiation. Only one well, well #16 containing IL-1-alpha,contained contracting rhythmically myocardial cells. Interestingly,these cells were and consistently found to be growing in associationwith underlying endothelial cells. FIGS. 12A and 12B are phase contrastphotographs of the cells in well #16. The arrowhead in FIG. 12A pointsto a beating myocardial cell. The arrowhead in FIG. 12B points to anendothelial ceil inducers adjacent to myocardial cells.

EXAMPLE 6 Screen for Agents that Induce Differentiation of Murine ESCells; Detection By ICC

Approximately 5,000 murine ES cells (strain J1) were plated in a 24 welltissue culture plate without LIF in 1.5 mL of DMEM Medium with 15% fetalbovine serum, added nonessential amino acids, 5×10⁻⁵ M2-mercaptoethanol, 2 mM L-glutamine, 100 ug/ml penicillin, and 100 ug/mlstreptomycin. The cells were allowed to differentiate in the presence ofFGF-4 and/or TGF-beta-1 (concentrations as in Example 2), in thepresence or absence of inducer fibroblasts, or in the presence orabsence of type I collagen and human plasma fibronectin (the wells wereprecoated by incubating for an hour with 10 ug/mL of the ECM proteins,and then removing and rinsing in PBS). The combinations of putativedifferentiation-inducing agents in each well are shown in Table 3 ofFIG. 13.

After incubating the cells for five days in the presence of the putativedifferentiation-inducing agents, the cells in the wells were assayed forexpression of cell type-associated genes by ICC. Primary antibodies todesmin, nestin, and GATA-4 were applied to the cells and visualized byfluorescence microscopy as described in Example 3 above. FIG. 14 showsimmunofluorescence from anti-desmin antibody bound to desmin, a markerof mesodermal cell lineages, in murine ES cells cultured in TGF-beta-1and FGF4 for five days on type I collagen and human plasma fibronectin.

The expression of cell type-associated genes such as GATA-4 and nestinby the murine ES cells in the wells that were induced to differentiatewas also detected by RT-PCR assay performed as described in Example 2(data not shown).

EXAMPLE 7 Screen with Murine Gene-Trap ES Cell Lines; Detection by X-galStaining and ICC

Cells of the murine gene trap ES cell lines K18E2 and M7H7 each have DNAencoding beta-galactosidase inserted as a marker gene in a genetic locusthat is activated when the cells differentiate. The DNA encodingbeta-galactosidase is inserted in-frame with correct orientation at asite such that it is expressed and beta-galactosidase is produced whenthe genetic locus in which it is inserted is activated. Accordingly, thebeta-galactosidase coding sequence operates as a marker permittingdetection of the differentiation of K18E2 and M7H7 ES cells. Thebeta-galactosidase marker DNA is inserted at different loci in K18E2 andM7H7 ES cells, and the sets of conditions that leads to activation ofthe marker gene are not the same for the two cell types. Thebeta-galactosidase marker in K18E2 ES cells is expressed in many earlydifferentiated cell lineages; the beta-galactosidase marker in M7H7cells is expressed in early mesoderm and retains expression inendothelial and hematopoietic pathways.

Cells of murine gene trap cell lines K18E2 were treated as described inExample 6 above and subsequently stained with X-gal to detect expressionof the marker beta-galactosidase gene. X-gal staining is generally wellknown in the art. Briefly, the cells were washed once with 0.1Mphosphate buffer, fixed at room temperature in 25% gluteraldehyde in0.1M phosphate buffer, washed again five times in phosphate buffer, andstained overnight at 37 degrees C. with X-gal stain. The pH of thebuffer was in the range of 7.0-8.0 depending on the cells used.

FIG. 15 shows the detection of X-gal staining of K18E2 ES cells thatwere cultured for five days on type I collagen and human plasmafibronectin in the presence of TGF-beta-1 and FGF-4. Detection ofexpression of the beta-galactosidase marker gene in cells derived fromthe K18E2 ES cells indicates that the cells were induced todifferentiate.

Expression of the beta-galactosidase marker gene in K18E2 and M7H7 EScells that were cultured in the presence of differentiation-inducingagents was also detected by ICC. FIG. 16 shows the detection ofbeta-galactosidase by ICC (using antibody to beta-galactosidase) in M7H7ES cells that were cultured for five days on type I collagen and humanplasma fibronectin in the presence of TGF-beta-1 and FGF-4. Cell nucleiwere co-visualized by DAPI staining. FIG. 17 shows the detection ofbeta-galactosidase by ICC in K18E2 ES cells that were cultured for fivedays on type I collagen and human plasma fibronectin in the presence ofFGF-4.

Using RT-PCR to detect expression, the beta-galactosidase marker gene inmurine gene trap ES cells was also shown to be activated when the cellswere induced to differentiate by other cells (data not shown).

EXAMPLE 8 Screen for Induction of Differentiation by Cell-CellInteractions

Murine gene trap K18E2 and M7H7 ES cells were plated in wells of a24-well tissue culture plate (5,000 to 20,000 cells/well) and wereallowed to differentiate in the presence of FGF-4 and TGF-beta-1,generally as described in Example 6 above, except that in some of thewells, the cells were plated onto a layer fibroblast mesenchymal inducercells. After incubation for five days, the cells were all transferred towells containing FGF-4 and TGF-beta-1 without inducer cells, and werecultured for an additional five days. Following this treatment,expression of the beta-galactosidase marker gene was detected by the ICCprotocol described in Example 3. The images in FIGS. 18, 19, 20 and 21are of labeling using monoclonal anti-β-galactosidase (G-6282Sigma-Aldrich, Inc.) primary antibody and anti-mouse IgM FITC conjugated(F9259 Sigma-Aldrich, Inc.) secondary antibody.

Results

FIG. 18 shows the presence of β-galactosidase in K18E2 cells that werecultured with FGF-4 and TGF-β1 on inducer fibroblasts for 5 days, thensub-cultured for an additional 5 days with FGF-4 and TGF-β1 alone. FIG.19 shows the presence of β-galactosidase in M7H7 cells that werecultured with FGF-4 and TGF-β1 on inducer fibroblasts for 5 days, thensub-cultured for an additional 5 days with FGF-4 and TGF-β1 alone. FIG.20 shows the presence of β-galactosidase in K18E2 cells that werecultured with FGF-4 and TGF-β1 in the absence of inducer fibroblasts,and then sub-cultured for 5 more days in same conditions. FIG. 21 showsthe presence of β-galactosidase in M7H7 cells that were cultured withFGF-4 and TGF-β1 in the absence of inducer fibroblasts, and thensub-cultured for 5 more days in same conditions.

The beta-galactosidase marker gene was expressed by both lines of genetrap stem cells cultured with FGF-4 and TGF-β1 on inducer fibroblasts,and also by the same stem cells cultured with FGF-4 and TGF-β1 alone.However, the beta-galactosidase marker gene was expressed by the M7H7cells cultured with FGF-4 and TGF-β1 on inducer fibroblastssignificantly more strongly than by the M7H7 cells that were culturedwith FGF-4 and TGF-β1 alone. The beta-galactosidase marker gene of M7H7is activated when the cells are induced to differentiate into cells ofthe mesodermal lineage, and are therefore useful for identifyingconditions that induce the stem cells to differentiate intohematopoietic cells. This example demonstrates the use of the inventionto identify cell-cell interactions between stem cells and inducerfibroblasts that operate to induce differentiation of stem cells intocells of the mesodermal lineage, e.g., for producing hematopoieticcells.

EXAMPLE 9 Directing Differentiation with Multi-Nodal Markers

This example demonstrates how multi-nodal markers can be used toidentify differentiated cell types.

Cell line A is a totipotent gene trap stem cell line with a gene trapmarker that is expressed when the cells are exposed to three differentsets of conditions that direct them to differentiate, respectively, intoheart, lung, and kidney.

Cell line B is a totipotent gene trap stem cell line with a differentgene trap marker that is expressed when the cells are exposed to threedifferent sets of conditions that direct them to differentiate,respectively, into lung, brain, and eye.

Cell Types in Which the Gene Trap Marker is Expressed

Cell line A Cell line B heart eye lung lung kidney brain

Screening is performed to identify a set of conditions that activatesthe marker in both cell lines; this set of conditions is expected todirect differentiation to lung.

EXAMPLE 10 Screening in Eggs

An array of avian eggs is used as the set of compartments in whichscreening for differentiation is performed. 10² to 10⁵ totipotent,nearly totipotent, or pluripotent stem cells; e.g., murine or primate EScells, are introduced into each egg. One or more putativedifferentiation-inducing agents; e.g., growth factors, cytokines, ECMcompounds, and/or inducer cells, are then added to the cells in each eggin various combinations and temporal sequences. The eggs are incubatedand activation of cell type-associated genes in the cells is detected byRT-PCR.

The assay can be performed using gene trap ES cells having gene trapmarkers that are activated when the stem cells differentiate intospecific cell types. Use of such cells permits two types of screening tobe performed. In one, an array of eggs is prepared with each eggcontaining the same type of gene trap stem cell, and a differentcombination of putative differentiation-inducing agents. In the other,an array of eggs is prepared with each egg containing stem cells havinga different gene trap marker that is activated when the cell is inducedto differentiate, and the same combination of putativedifferentiation-inducing agents. The first assay is a screen to identifyagents or conditions that direct differentiation of stem cells into aspecific cell type. The second assay identifies cell type-associatedmarkers that are activated by a particular set of putativedifferentiation-inducing agents.

EXAMPLE 11 Screens Utilizing Lineage Tracers Introduced by Site-SpecificRecombination

For efficient detection of the activation of a genetic locus that isonly transiently activated at a step or “node” in the branching pathwayleading to differentiation to a desired specific cell type, gene trapstem cells can be made by inserting two coding sequences into the genomeof the stem cell:

-   -   (i) a sequence encoding a recombinase that is inserted into the        locus in-frame with correct orientation at a site such that it        is expressed and recombinase is produced when the genetic locus        in which it is inserted is activated; and    -   (ii) a sequence encoding a marker protein that is disrupted by a        nucleotide sequence with flanking recombinase sites that is        excised by the recombinase to generate an undisrupted marker        gene. This sequence can be inserted into a genetic locus that is        constitutively active, or into the same locus as the recombinase        DNA.

When the genetic locus in which the recombinase DNA is inserted isactivated, recombinase is synthesized and catalyzes excision of thedisrupting sequence from the marker DNA sequence, permitting detectionof the marker in the differentiated cell. When transcription of themarker DNA is under control of a constitutively active promoter, themarker can be detected even when the locus in which the recombinase DNAis inserted is a transiently activated locus that subsequently becomesdeactivated (see Zinyk et al., Curr. Biol. (1998) 8:665-668; Dymecki etal., Dev. Biol. (1998) 201:57-65, each incorporated by reference in itsentirety). For example, the recombinase systems such as that of the λintegrase family can be used to implement this method. The cre-loxP andFLO-FRT systems allow the activation or inactivation of target sequencesthat operate as permanent markers in the genomes of cells having passedcertain points in development. The use of these systems in fate mappingcells in animal development is well known in the art; however, the useof recombinase-mediated cell fate marking for the in vitro screening ofstem cell differentiation has not been described. Current fate mappingtechniques utilize two components: 1) a recombinase animal thatexpresses the recombinase (Cre or FLP) in a gene-specific manner, and 2)the indicator animal that has a transgene activated in the presence ofthe recombinase in a permanent fashion e.g., such that β-gal isexpressed in this and all cells derived from such a cell regardless oftheir differentiated state. This recombinase can be introduced into EScells in gene trap vectors as described above, and the recombinant EScells can be used to produce an assortment of individual recombinasemice that can provide a random assortment of gametes harboring many genetrapped recombinase genes. These gametes (sperm or eggs) can then beused with the complementary gamete from the indicator animal to produceembryos, embryoid bodies, or stem cells that leave a permanent marker ofhaving passed a given point in the developmental tree. Suchlineage-tracing stem cells have particular utility when the gene ofinterest is only transiently expressed and therefore difficult todetect. Libraries of stem cells in which such recombinase-based markersare randomly inserted may be made and screened to identify cell typeassociated gene trap markers. Alternatively, libraries of stem cells inwhich such recombinase-based markers are targeted to specific loci areuseful in the screening assay of the present invention for determiningthe conditions under which stem cells are induced to express celltype-associated genes and differentiate into a particular cell type.

1. A method for evaluating the differentiation of totipotent, nearlytotipotent, or pluripotent stem cells, or cells therefrom, in responseto one or more chemical or biological agents or physical conditions,comprising: (a) separating individual totipotent, nearly totipotent, orpluripotent stem cells, or cells therefrom, or groups of such cells, inculture medium into one or a plurality of separate wells which may beopen or closed, which wells may be in the same or different apparatus;(b) exposing said separate wells of cells to one or more putativedifferentiation-inducing conditions simultaneously or sequentially; and(c) screening said individual cells or groups of cells to detect markersof differentiation of said individual cells or groups of cells, whereinthe markers are indicative of differentiation to myocardial cells. 2.(canceled)
 3. The method of claim 1, wherein said nearly totipotent, orpluripotent stem cells, or cells therefrom, are selected from the groupconsisting of human cells, primate cells, bovine cells, porcine cells,murine cells, rat cells, sheep cells, canine and feline cells.
 4. Themethod of claim 1, wherein said one or more putativedifferentiation-inducing conditions are selected from the groupconsisting of growth factors, cytokines, tissue extracts, nucleic acids,factors involved in cell-to-cell interactions, adhesion molecules andextracellular matrix components, extracts of extracellular componentsfrom tissue, media components, environmental conditions, and livingcells that induce differentiation by cell-cell interactions.
 5. Themethod of claim 4, wherein said growth factors and cytokines areselected from the group consisting of the Fibroblast Growth Factorfamily of proteins (FGF1-23), the Tumor necrosis factor (TNF)superfamily (TNFSF), the insulin-like growth factor family IGF-1 andtheir binding proteins, the matrix metalloproteinases PDGF, Flt-3ligand, Fas Ligand, B7-1(CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 Ralpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, GDNF, G-CSF,GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR beta,Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF,TGF-alpha, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sRalpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF,Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin,IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1,GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5,MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin,Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha(EGF Domain), TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4,BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14,CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4),VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2(KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19,Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9,NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5,IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D,Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, ActivinRIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta (IIIb),DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma RI,IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc),FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha(IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin(CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4,Osteoprotegerin (OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II,IL-18 R, IL-12 R beta 1, Dtk, LBP, SDF-1 alpha (PBSF)/CXCL12(synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P),ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1), hedgehog family ofproteins, Interleukin-10, Epidermal Growth Factor, Heregulin, HER4,Heparin Binding Epidermal Growth Factor, bFGF, MIP-18, MIP-2, MCP-1,MCP-5, NGF, NGF-B, leptin, Interferon A, Interferon A/D, Interferon B,Interferon Inducible Protein-10, Insulin Like Growth Factor-II,IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil Chemoattractant2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine InducedNeutrophil Chemoattractant 1, Cytokine Responsive Gene-2, and anyfragment thereof and their neutralizing antibodies.
 6. The method ofclaim 4, wherein said factors involved in cell-cell interactions areselected from the group consisting of the ADAM (A Disintegrin andMetalloproteinase) family of proteins including ADAM 1,2,3A, 3B, 4-31and TS1-9, ADAMTSs (ADAMs with thrombospondin motifs), Reprolysins,metzincins, zincins, and zinc metalloproteinases and their neutralizingantibodies.
 7. The method of claim 4, wherein said adhesion moleculesare selected from the group consisting of Ig superfamily CAM's,Integrins, Cadherins and Selectins and their neutralizing antibodies. 8.The method of claim 4, wherein said nucleic acids that may be tested arethose that encode or block by antisense, ribozyme activity, or RNAinterference with transcription factors that are involved in regulatinggene expression during differentiation, genes for growth factors,cytokines, and extracellular matrix components, or other molecularactivities that regulate differentiation.
 9. The method of claim 4wherein said cell-cell interactions include placing the cells beingassayed in cell-cell contact with cells of another differentiated celltype, or in the presence of media conditioned by cells of anotherdifferentiated cell type.
 10. The method of claim 4 wherein said tissueextracts comprise materials derived from early stage embryos, fetuses,or adult tissues. 11-86. (canceled)
 87. The method of claim 4,comprising exposing the stem cells to endothelial cells.